Method for the production and purification of adenoviral vectors

ABSTRACT

The present invention addresses the need to improve the yields of viral vectors when grown in cell culture systems. In particular, it has been demonstrated that for adenovirus, the use of low-medium perfusion rates in an attached cell culture system provides for improved yields. In other embodiments, the inventors have shown that there is improved Ad-p53 production with cells grown in serum-free conditions, and in particular in serum-free suspension culture. Also important to the increase of yields is the use of detergent lysis. Combination of these aspects of the invention permits purification of virus by a single chromatography step that results in purified virus of the same quality as preparations from double CsCl banding using an ultracentrifuge.

BACKGROUND OF THE INVENTION

The present application is a continuation-in-part of co-pending U.S.Provisional Patent Application Ser. No. 60/031,329 filed Nov. 20, 1997.The entire text of the above-referenced disclosure is specificallyincorporated by reference herein without disclaimer.

1. Field of the Invention

The present invention relates generally to the fields of cell cultureand virus production. More particularly, it concerns improved methodsfor the culturing of mammalian cells, infection of those cells withadenovirus and the production of infectious adenovirus particlestherefrom.

2. Description of Related Art

Adenoviral vectors, which express therapeutic proteins, are currentlybeing evaluated in the clinic for the treatment of a variety of cancerindications, including lung and head and neck cancers. As the clinicaltrials progress, the demand for clinical grade adenoviral vectors isincreasing dramatically. The projected annual demand for a 300 patientclinical trial could reach approximately 6×10¹⁴ PFU.

Traditionally, adenoviruses are produced in commercially availabletissue culture flasks or “cellfactories.” Virus infected cells areharvested and freeze-thawed to release the viruses from the cells in theform of crude cell lysate. The produced crude cell lysate (CCL) is thenpurified by double CsCl gradient ultracentrifugation. The typicallyreported virus yield from 100 single tray cellfactories is about 6×10¹²PFU. Clearly, it becomes unfeasible to produce the required amount ofvirus using this traditional process. New scaleable and validatableproduction and purification processes have to be developed to meet theincreasing demand.

The purification throughput of CsCl gradient ultracentrifugation is solimited that it cannot meet the demand for adenoviral vectors for genetherapy applications. Therefore, in order to achieve large scaleadenoviral vector production, purification methods other than CsClgradient ultracentrifugation have to be developed. Reports on thechromatographic purification of viruses are very limited, despite thewide application of chromatography for the purification of recombinantproteins. Size exclusion, ion exchange and affinity chromatography havebeen evaluated for the purification of retroviruses, tick-borneencephalitis virus, and plant viruses with varying degrees of success(Crooks, et al., 1990; Aboud, et al., 1982; McGrath et al., 1978, Smithand Lee, 1978; O'Neil and Balkovic, 1993). Even less research has beendone on the chromatographic purification of adenovirus. This lack ofresearch activity may be partially attributable to the existence of theeffective, albeit non-scalable, CsCl gradient ultracentrifugationpurification method for adenoviruses.

Recently, Huyghe et al. (1996) reported adenoviral vector purificationusing ion exchange chromatography in conjunction with metal chelateaffinity chromatography. Virus purity similar to that from CsCl gradientultracentrifugation was reported. Unfortunately, only 23% of virus wasrecovered after the double column purification process. Process factorsthat contribute to this low virus recovery are the freeze/thaw steputilized by the authors to lyse cells in order to release the virus fromthe cells and the two column purification procedure.

Clearly, there is a demand for an effective and scaleable method ofadenoviral vector production that will recover a high yield of productto meet the ever increasing demand for such products.

SUMMARY OF THE INVENTION

The present invention describes a new process for the production andpurification of adenovirus. This new production process offers not onlyscalability and validatability but also virus purity comparable to thatachieved using CsCl gradient ultracentrifugation.

Thus the present invention provides a method for producing an adenoviruscomprising growing host cells in media at a low perfusion rate,infecting the host cells with an adenovirus, harvesting and lysing thehost cells to produce a crude cell lysate, concentrating the crude celllysate, exchanging buffer of crude cell lysate, and reducing theconcentration of contaminating nucleic acids in the crude cell lysate.

In particular embodiments, the method further comprises isolating anadenoviral particle from the lysate using chromatography. In certainembodiments, the isolating consists essentially of a singlechromatography step. In other embodiments, the chromatography step ision exchange chromatography. In particularly preferred embodiments, theion exchange chromatography is carried out at a pH range of betweenabout 7.0 and about 10.0. In more preferred embodiments, the ionexchange chromatography is anion exchange chromatography. In certainembodiments the anion exchange chromatography utilizes DEAE, TMAE, QAE,or PEI. In other preferred embodiments, the anion exchangechromatography utilizes Toyopearl Super Q 650M, MonoQ, Source Q orFractogel TMAE.

In certain embodiments of the present invention the glucoseconcentration in the media is maintained between about 0.7 and about 1.7g/L. In certain other embodiments, the exchanging buffer involves adiafiltration step.

In preferred embodiments of the present invention, the adenoviruscomprises an adenoviral vector encoding an exogenous gene construct. Incertain such embodiments, the gene construct is operatively linked to apromoter. In particular embodiments, the promoter is SV40 IE, RSV LTR,β-actin or CMV IE, adenovirus major late, polyoma F9-1, or tyrosinase.In particular embodiments of the present invention, the adenovirus is areplication-incompetent adenovirus. In other embodiments, the adenovirusis lacking at least a portion of the E1-region. In certain aspects, theadenovirus is lacking at least a portion of the E1A and/or E1B region.In other embodiments, the host cells are capable of complementingreplication. In particularly preferred embodiments, the host cells are293 cells.

In preferred a embodiment of the present invention it is contemplatedthat the exogenous gene construct encodes a therapeutic gene. Forexample, the therapeutic gene may encode antisense ras, antisense myc,antisense raf, antisense erb, antisense src, antisense fms, antisensejun, antisense trk, antisense ret, antisense gsp, antisense hst,antisense bcl antisense abl, Rb, CFTR, p16, p21, p27, p57, p73, C-CAM,APC, CTS-1, zac1, scFV ras, DCC, NF-1, NF-2, WT-1, MEN-I, MEN-11, BRCA1,VHL, MMAC1, FCC, MCC, BRCA2, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-8, IL-9, IL-10, IL-11 IL-12, GM-CSF G-CSF, thymidine kinase or p53.

In certain aspects of the present invention, the cells may be harvestedand lysed ex situ using a hypotonic solution, hypertonic solution,freeze-thaw, sonication, impinging jet, microfluidization or adetergent. In other aspects, the cells are harvested and lysed in situusing a hypotonic solution, hypertonic solution, or a detergent. As usedherein the term “in situ” refers to the cells being located within thetissue culture apparatus for example CellCube™ and “ex situ” refers tothe cells being removed from the tissue culture apparatus.

In particular embodiments, the cells are lysed and harvested usingdetergent. In preferred embodiments the detergent may be Thesit®,NP-40®, Tween-20®, Brij-58®, Triton X®-100 or octyl glucoside. In otheraspects of the present invention lysis is achieved through autolysis ofinfected cells. In certain other aspects of the present invention thecell lysate is treated with Benzonase®, or Pulmozyme®.

In particular embodiments, the method further comprises a concentrationstep employing membrane filtration. In particular embodiments, thefiltration is tangential flow filtration. In preferred embodiments, thefiltration may utilize a 100 to 300K NMWC, regenerated cellulose, orpolyether sulfone membrane.

The present invention also provides an adenovirus produced according toa process comprising the steps of growing host cells in media at a lowperfusion rate, infecting the host cells with an adenovirus, harvestingand lysing the host cells to produce a crude cell lysate, concentratingthe crude cell lysate, exchanging buffer of crude cell lysate, andreducing the concentration of contaminating nucleic acids in the crudecell lysate.

Other aspects of the present invention provide a method for thepurification of an adenovirus comprising growing host cells, infectingthe host cells with an adenovirus, harvesting and lysing the host cellsby contacting the cells with a detergent to produce a crude cell lysate,concentrating the crude cell lysate, exchanging buffer of crude celllysate, and reducing the concentration of contaminating nucleic acids inthe crude cell lysate.

In particular embodiments; the detergent may be Thesit®, NP-40®,Tween-20®, Brij-580, Triton X-100® or octyl glucoside. In moreparticular embodiments the detergent is present in the lysis solution ata concentration of about 1% (w/v).

In other aspects of the present invention there is provided anadenovirus produced according to a process comprising the steps ofgrowing host cells, infecting the host cells with an adenovirus,harvesting and lysing the host cells by contacting the cells with adetergent to produce a crude cell lysate, concentrating the crude celllysate, exchanging buffer of crude cell lysate, and reducing theconcentration of contaminating nucleic acids in the crude cell lysate.

In yet another embodiment, the present invention provides a method forthe purification of an adenovirus comprising the steps of growing hostcells in serum-free media; infecting said host cells with an adenovirus;harvesting and lysing said host cells to produce a crude cell lysate;concentrating said crude cell lysate; exchanging buffer of crude celllysate; and reducing the concentration of contaminating nucleic acids insaid crude cell lysate. In preferred embodiments, the cells may be grownindependently as a cell suspension culture or as an anchorage-dependentculture.

In particular embodiments, the host cells are adapted for growth inserum-free media. In more preferred embodiments, the adaptation forgrowth in serum-free media comprises a sequential decrease in the fetalbovine serum content of the growth media. More particularly, theserum-free media comprises a fetal bovine serum content of less than0.03% v/v.

In other embodiments, the method further comprises isolating anadenoviral particle from said lysate using chromatography. In preferredembodiments, the isolating consists essentially of a singlechromatography step. More particularly, the chromatography step is ionexchange chromatography.

Also contemplated by the present invention is an adenovirus producedaccording to a process comprising the steps of growing host cells inserum-free media; infecting said host cells with an adenovirus;harvesting and lysing said host cells to produce a crude cell lysate;concentrating said crude cell lysate; exchanging buffer of crude celllysate; and reducing the concentration of contaminating nucleic acids insaid crude cell lysate.

The present invention further provides a 293 host cell adapted forgrowth in serum-free media. In certain aspects, the adaptation forgrowth in serum-free media comprises a sequential decrease in the fetalbovine serum content of the growth media. In particular embodiments, thecell is adapted for growth in suspension culture. In particularembodiments, the cells of the present invention are designated IT293SFcells. These cells were deposited with the American Tissue CultureCollection (ATCC) in order to meet the requirements of the BudapestTreaty on the international recognition of deposits of microorganismsfor the purposes of patent procedure. The cells were deposited by Dr.Shuyuan Zhang on behalf of Introgen Therapeutics, Inc. (Houston, Tx.),on Nov. 17, 1997. IT293SF cell line is derived from an adaptation of 293cell line into serum free suspension culture as described herein. Thecells may be cultured in IS 293 serum-free media (Irvine Scientific.Santa Ana, Calif.) supplemented with 100 mg/L heparin and 0.1% pluronicF-68, and are permissive to human adenovirus infection.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1A and FIG. 1B. HPLC profiles of the viral solutions fromproduction runs using medium perfusion rates characterized as “high”(FIG. 1A) and “low” (FIG. 1B).

FIG. 2. The HPLC profile of crude cell lysate (CCL) from CellCube™(solid line A₂₆₀; dotted line A₂₈₀).

FIG. 3A, FIG. 3B, FIG. 3C, FIG. 3D and FIG. 3E. The HPLC profiles oflysis solutions from CellCube™ using different detergents. FIG. 3AThesit®. FIG. 3B Triton®X-100. FIG. 3C. NP-40′. FIG. 3D. Brij®80. FIG.3E. Tween®20. Detergent concentration: 1% (w/v) lysis temperature: roomtemperature. (solid line A₂₆₀; dotted line A₂₈₀).

FIG. 4A and FIG. 4B. The HPLC profiles of virus solution before (FIG.4A) and after (FIG. 4B) Benzonase treatment. (solid line A₂₆₀; dottedline A₂₈₀).

FIG. 5. The HPLC profile of virus solution after Benzonase treatment inthe presence of 1M NaCl. (solid line A₂₆₀; dotted line A₂₈₀).

FIG. 6. Purification of AdCMVp53 virus under buffer A condition of 20 mMTris+1 mM MgCl₂+0.2M NaCl, pH=7.5.

FIG. 7. Purification of AdCMVp53 virus under buffer A condition of 20 mMTris+1 mM MgCl₂+0.2M NaCl, pH=9.0.

FIG. 8A, FIG. 8B, and FIG. 8C. HPLC analysis of fractions obtainedduring purification FIG. 8A fraction 3. FIG. 8B fraction 4, FIG. 8Cfraction 8. (solid line A₂₆₀; dotted line A₂₈₀).

FIG. 9. Purification of AdCMVp53 virus under buffer A condition of 20 mMTris+1 mM MgCl₂+0.3M NaCl, pH=9.

FIG. 10A, FIG. 10B, FIG. 10C, FIG. 10D and FIG. 10E. HPLC analysis ofcrude virus fractions obtained during purification and CsCl gradientpurified virus. FIG. 10A Crude virus solution. FIG. 10B Flow through.FIG. 10C. Peak number 1. FIG. 10D. Peak number 2. FIG. 10E. CsClpurified virus. (solid line A₂₆₀; dotted line A₂₈₀).

FIG. 11. HPLC purification profile from a 5 cm id column.

FIG. 12. The major adenovirus structure proteins detected on SDS-PAGE.

FIG. 13. The BSA concentration in the purified virus as detected levelof the western blot assay.

FIG. 14. The chromatogram for the crude cell lysate material generatedfrom the CellCube™.

FIG. 15. The elution profile of treated virus solution purified usingthe method of the present invention using Toyopearl SuperQ resin.

FIG. 16A and FIG. 16B. HPLC analysis of virus fraction from purificationprotocol. FIG. 16A HPLC profiles of virus fraction from firstpurification step. FIG. 16B HPLC profiles of virus fraction from secondpurification. (solid line A₂₆₀; dotted line A₂₈₀).

FIG. 17. Purification of 1% Tween® harvest virus solution under lowmedium perfusion rate.

FIG. 18. HPLC analysis of the virus fraction produced under low mediumperfusion rate.

FIG. 19A, FIG. 19B and FIG. 19C. Analysis of column purified virus. FIG.19A SDS-PAGE analysis. FIG. 19B Western blot for BSA. FIG. 19C nucleicacid slot blot to determine the contaminating nucleic acidconcentration.

FIG. 20A, FIG. 20B, FIG. 20C, FIG. 20D, FIG. 20E and FIG. 20F. Capacitystudy of the Toyopearl SuperQ 650M resin. FIG. 20A Flow through fromloading ratio of 1:1. FIG. 20B. Purified virus from loading ratio of1:1. FIG. 20C Flow through of loading ratio of 2:1. FIG. 20D. Purifiedvirus from the loading ratio of 2:1. FIG. 20E Flow through from loadingratio of 3:1. FIG. 20F. Purified virus from the loading ratio of 3:1.(solid line A₂₆₀; dotted line A₂₈₀).

FIG. 21. Isopycnic CsCl ultracentrifugation column purified virus.

FIG. 22. The HPLC profiles of intact viruses present in the columnpurified virus. A. Intact virus B. Defective virus. (solid line A₂₆₀;dotted line A₂₈₀).

FIG. 23. A production and purification flow chart for AdCMVp53

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

It has been shown that adenoviral vectors can successfully be used ineukaryotic gene expression and vaccine development. Recently, animalstudies have demonstrated that recombinant adenovirus could be used forgene therapy. Successful studies in administering recombinant adenovirusto different tissues have proven the effectiveness of adenoviral vectorsin therapy. This success has led to the use of such vectors in humanclinical trials. There now is an increased demand for the production ofadenoviral vectors to be used in various therapies. The techniquescurrently available are insufficient to meet such a demand. The presentinvention provides methods for the production of large amounts ofadenovirus for use in such therapies.

The present invention involves a process that has been developed for theproduction and purification of a replication deficient recombinantadenovirus. The production process is based on the use of a Cellcube™bioreactor for cell growth and virus production. It was found that agiven perfusion rate, used during cell growth and the virus productionphases of culturing, has a significant effect on the downstreampurification of the virus. More specifically, a low to medium perfusionrate improves virus production. In addition, lysis solution composed ofbuffered detergent, used to lyse cells in the Cellcube™ at the end ofvirus production phase, also improves the process. With these twoadvantages, the harvested crude virus solution can be purified using asingle ion exchange chromatography run, afterconcentration/diafiltration and nuclease treatment to reduce thecontaminating nucleic acid concentration in the crude virus solution.The column purified virus has equivalent purity relative to that ofdouble CsCl gradient purified virus. The total process recovery of thevirus product is 70%±10%. This is a significant improvement over theresults reported by Huyghe et al. (1996). Compared to double CsClgradient ultracentrifugation, column purification has the advantage ofbeing more consistent, scaleable, validatable, faster and lessexpensive. This new process represents a significant improvement in thetechnology for manufacturing of adenoviral vectors for gene therapy.

Therefore, the present invention is designed to take advantage of theseimprovements in large scale culturing systems and purification for thepurpose of producing and purifying adenoviral vectors. The variouscomponents for such a system, and methods of producing adenovirustherewith, are set forth in detail below.

1. HOST CELLS

A) Cells

In a preferred embodiment, the generation and propagation of theadenoviral vectors depend on a unique helper cell line, designated 293,which was transformed from human embryonic kidney cells by Adenovirusserotype 5 (Ad5) DNA fragments and constitutively expresses E1 proteins(Graham et al., 1977). Since the E3 region is dispensable from the Adgenome (Jones and Shenk, 1978), the current Ad vectors, with the help of293 cells, carry foreign DNA in either the E1, the E3 or both regions(Graham and Prevec, 1991; Bett et al., 1994).

A first aspect of the present invention is the recombinant cell lineswhich express part of the adenoviral genome. These cells lines arecapable of supporting replication of adenovirus recombinant vectors andhelper viruses having defects in certain adenoviral genes, i.e., are“permissive” for growth of these viruses and vectors. The recombinantcell also is referred to as a helper cell because of the ability tocomplement defects in, and support replication of,replication-incompetent adenoviral vectors. The prototype for anadenoviral helper cell is the 293 cell line, which contains theadenoviral E1 region. 293 cells support the replication of adenoviralvectors lacking E1 functions by providing in trans the E1-activeelements necessary for replication.

Helper cells according to the present invention are derived from amammalian cell and, preferably, from a primate cell such as humanembryonic kidney cell. Although various primate cells are preferred andhuman or even human embryonic kidney cells are most preferred, any typeof cell that is capable of supporting replication of the virus would beacceptable in the practice of the invention. Other cell types mightinclude, but are not limited to Vero cells, CHO cells or any eukaryoticcells for which tissue culture techniques are established as long as thecells are adenovirus permissive. The term “adenovirus permissive” meansthat the adenovirus or adenoviral vector is able to complete the entireintracellular virus life cycle within the cellular environment.

The helper cell may be derived from an existing cell line, e.g., from a293 cell line, or developed de novo. Such helper cells express theadenoviral genes necessary to complement in trans deletions in anadenoviral genome or which supports replication of an otherwisedefective adenoviral vector, such as the E1, E2, E4, E5 and latefunctions. A particular portion of the adenovirus genome, the E1 region,has already been used to generate complementing cell lines. Whetherintegrated or episomal, portions of the adenovirus genome lacking aviral origin of replication, when introduced into a cell line, will notreplicate even when the cell is superinfected with wild-type adenovirus.In addition, because the transcription of the major late unit is afterviral DNA replication, the late functions of adenovirus cannot beexpressed sufficiently from a cell line. Thus, the E2 regions, whichoverlap with late functions (L1-5), will be provided by helper virusesand not by the cell line. Typically, a cell line according to thepresent invention will express E1 and/or E4.

As used herein, the term “recombinant” cell is intended to refer to acell into which a gene, such as a gene from the adenoviral genome orfrom another cell, has been introduced. Therefore, recombinant cells aredistinguishable from naturally-occurring cells which do not contain arecombinantly-introduced gene. Recombinant cells are thus cells having agene or genes introduced through “the hand of man.”

Replication is determined by contacting a layer of uninfected cells, orcells infected with one or more helper viruses, with virus particles,followed by incubation of the cells. The formation of viral plaques, orcell free areas in the cell layer, is the result of cell lysis caused bythe expression of certain viral products. Cell lysis is indicative ofviral replication.

Examples of other useful mammalian cell lines that may be used with areplication competent virus or converted into complementing host cellsfor use with replication deficient virus are Vero and HeLa cells andcell lines of Chinese hamster ovary, WI 38, BHK, COS-7, HepG2, 3T3, RINand MDCK cells.

B) Growth in Selection Media

In certain embodiments, it may be useful to employ selection systemsthat preclude growth of undesirable cells. This may be accomplished byvirtue of permanently transforming a cell line with a selectable markeror by transducing or infecting a cell line with a viral vector thatencodes a selectable marker. In either situation, culture of thetransformed/transduced cell with an appropriate drug or selectivecompound will result in the enhancement, in the cell population, ofthose cells carrying the marker.

Examples of markers include, but are not limited to, HSV thymidinekinase, hypoxanthine-guanine phosphoribosyltransferase and adeninephosphoribosyltransferase genes, in tk-; hgprt- or aprt- cells,respectively. Also, anti-metabolite resistance can be used as the basisof selection for dhfr, that confers resistance to methotrexate; gpt,that confers resistance to mycophenolic acid; neo, that confersresistance to the aminoglycoside G418; and hygro, that confersresistance to hygromycin.

C. Growth in Serum Weaning

Serum weaning adaptation of anchorage-dependent cells into serum-freesuspension cultures have been used for the production of recombinantproteins (Berg, 1993) and viral vaccines (Perrin, 1995). There have beenfew reports on the adaptation of 293A cells into serum-free suspensioncultures until recently. Gilbert reported the adaptation of 293A cellsinto serum-free suspension cultures for adenovirus and recombinantprotein production (Gilbert, 1996). Similar adaptation method had beenused for the adaptation of A549 cells into serum-free suspension culturefor adenovirus production (Morris et al., 1996). Cell-specific virusyields in the adapted suspension cells, however, are about 5-10-foldlower than those achieved in the parental attached cells.

Using the similar serum weaning procedure, the inventors havesuccessfully adapted the 293A cells into serum-free suspension culture(293SF cells). In this procedure, the 293 cells were adapted to acommercially available 293 media by sequentially lowering down the FBSconcentration in T-flasks. Briefly, the initial serum concentration inthe media was approximately 10% FBS DMEM media in T-75 flask and thecells were adapted to serum-free IS 293 media in T-flasks by loweringdown the FBS concentration in the media sequentially. After 6 passagesin T-75 flasks the FBS % was estimated to be about 0.019% and the 293cells. The cells were subcultured two more times in the T flasks beforethey were transferred to spinner flasks. The results described hereinbelow show that cells grow satisfactorily in the serum-free medium(IS293 medium, Irvine Scientific, Santa Ana, Calif.). Average doublingtime of the cells were 18-24 h achieving stationary cell concentrationsin the order of 4-10×10⁶ cells/ml without medium exchange.

D. Adaptation of Cells for Suspension Culture

Two methodologies have been used to adapt 293 cells into suspensioncultures. Graham adapted 293A cells into suspension culture (293N35cells) by 3 serial passages in nude mice (Graham, 1987). The suspension293N35 cells were found to be capable of supporting El⁻ adenoviralvectors. However, Garnier et al. (1994) observed that the 293N35 cellshad a relatively long initial lag phase in suspension, a low growthrate, and a strong tendency to clump.

The second method that has been used is a gradual adaptation of 293Acells into suspension growth (Cold Spring Harbor Laboratories, 293Scells). Garnier et al. (1994) reported the use of 293S cells forproduction of recombinant proteins from adenoviral vectors. The authorsfound that 293S cells were much less clumpy in calcium-free media and afresh medium exchange at the time of virus infection could significantlyincrease the protein production. It was found that glucose was thelimiting factor in culture without medium exchange.

In the present invention, the 293 cells adapted for growth in serum-freeconditions were adapted into a suspension culture. The cells weretransferred in a serum-free 250 mL spinner suspension culture (100 mLworking volume) for the suspension culture at an initial cell density ofbetween about 1.18E+5 vc/mL and about 5.22E+5 vc/mL. The media may besupplemented with heparin to prevent aggregation of cells. This cellculture systems allows for some increase of cell density whilst cellviability is maintained. Once these cells are growing in culture, theycells are subcultured in the spinner flasks approximately 7 morepassages. It may be noted that the doubling time of the cells isprogressively reduced until at the end of the successive passages thedoubling time is about 1.3 day, i.e. comparable to 1.2 day of the cellsin 10% FBS media in the attached cell culture. In the serum-free IS 293media supplemented with heparin almost all the cells existed asindividual cells not forming aggregates of cells in the suspensionculture.

2. CELL CULTURE SYSTEMS

The ability to produce infectious viral vectors is increasinglyimportant to the pharmaceutical industry, especially in the context ofgene therapy. Over the last decade, advances in biotechnology have ledto the production of a number of important viral vectors that havepotential uses as therapies, vaccines and protein production machines.The use of viral vectors in mammalian cultures has advantages overproteins produced in bacterial or other lower lifeform hosts in theirability to post-translationally process complex protein structures suchas disulfide-dependent folding and glycosylation.

Development of cell culture for production of virus vectors has beengreatly aided by the development in molecular biology of techniques fordesign and construction of vector systems highly efficient in mammaliancell cultures, a battery of useful selection markers, gene amplificationschemes and a more comprehensive understanding of the biochemical andcellular mechanisms involved in procuring the final biologically-activemolecule from the introduced vector.

Frequently, factors which affect the downstream (in this case, beyondthe cell lysis) side of manufacturing scale-up were not consideredbefore selecting the cell line as the host for the expression system.Also, development of bioreactor systems capable of sustaining very highdensity cultures for prolonged periods of time have not lived up to theincreasing demand for increased production at lower costs.

The present invention will take advantage of the recently availablebioreactor technology. Growing cells according to the present inventionin a bioreactor allows for large scale production of fullybiologically-active cells capable of being infected by the adenoviralvectors of the present invention. By operating the system at a lowperfusion rate and applying a different scheme for purification of theinfecting particles, the invention provides a purification strategy thatis easily scaleable to produce large quantities of highly purifiedproduct.

Bioreactors have been widely used for the production of biologicalproducts from both suspension and anchorage dependent animal cellcultures. The most widely used producer cells for adenoviral vectorproduction are anchorage dependent human embryonic kidney cells (293cells). Bioreactors to be developed for adenoviral vector productionshould have the characteristic of high volume-specific culture surfacearea in order to achieve high producer cell density and high virusyield. Microcarrier cell culture in stirred tank bioreactor providesvery high volume-specific culture surface area and has been used for theproduction of viral vaccines (Griffiths, 1986). Furthermore, stirredtank bioreactors have industrially been proven to be scaleable. Themultiplate Cellcube™ cell culture system manufactured by Corning-Costaralso offers a very high volume-specific culture surface area. Cells growon both sides of the culture plates hermetically sealed together in theshape of a compact cube. Unlike stirred tank bioreactors, the Cellcube™culture unit is disposable. This is very desirable at the early stageproduction of clinical product because of the reduced capitalexpenditure, quality control and quality assurance costs associated withdisposable systems. In consideration of the advantages offered by thedifferent systems, both the stirred tank bioreactor and the Cellcube™system were evaluated for the production of adenovirus.

A) Anchorage-Dependent Versus Non-Anchorage-Dependent Cultures.

Animal and human cells can be propagated in vitro in two modes: asnon-anchorage dependent cells growing freely in suspension throughoutthe bulk of the culture; or as anchorage-dependent cells requiringattachment to a solid substrate for their propagation (i.e., a monolayertype of cell growth).

Non-anchorage dependent or suspension cultures from continuousestablished cell lines are the most widely used means of large scaleproduction of cells and cell products. Large scale suspension culturebased on microbial (bacterial and yeast) fermentation technology hasclear advantages for the manufacturing of mammalian cell products. Theprocesses are relatively simple to operate and straightforward to scaleup. Homogeneous conditions can be provided in the reactor which allowsfor precise monitoring and control of temperature, dissolved oxygen, andpH, and ensure that representative samples of the culture can be taken.

However, suspension cultured cells cannot always be used in theproduction of biologicals. Suspension cultures are still considered tohave tumorigenic potential and thus their use as substrates forproduction put limits on the use of the resulting products in human andveterinary applications (Petricciani, 1985; Larsson, 1987). Virusespropagated in suspension cultures as opposed to anchorage-dependentcultures can sometimes cause rapid changes in viral markers, leading toreduced immunogenicity (Bahnemann, 1980). Finally, sometimes evenrecombinant cell lines can secrete considerably higher amounts ofproducts when propagated as anchorage-dependent cultures as comparedwith the same cell line in suspension (Nilsson and Mosbach, 1987). Forthese reasons, different types of anchorage-dependent cells are usedextensively in the production of different biological products.

B) Reactors and Processes for Suspension.

Large scale suspension culture of mammalian cultures in stirred tankswas undertaken. The instrumentation and controls for bioreactorsadapted, along with the design of the fermentors, from related microbialapplications. However, acknowledging the increased demand forcontamination control in the slower growing mammalian cultures, improvedaseptic designs were quickly implemented, improving dependability ofthese reactors. Instrumentation and controls are basically the same asfound in other fermentors and include agitation, temperature, dissolvedoxygen, and pH controls. More advanced probes and autoanalyzers foron-line and off-line measurements of turbidity (a function of particlespresent), capacitance (a function of viable cells present),glucose/lactate, carbonate/bicarbonate and carbon dioxide are available.Maximum cell densities obtainable in suspension cultures are relativelylow at about 2-4×10⁶ cells/ml of medium (which is less than 1 mg drycell weight per ml), well below the numbers achieved in microbialfermentation.

Two suspension culture reactor designs are most widely used in theindustry due to their simplicity and robustness of operation—the stirredreactor and the airlift reactor. The stirred reactor design hassuccessfully been used on a scale of 8000 liter capacity for theproduction of interferon (Phillips et al., 1985; Mizrahi, 1983). Cellsare grown in a stainless steel tank with a height-to-diameter ratio of1:1 to 3:1. The culture is usually mixed with one or more agitators,based on bladed disks or marine propeller patterns. Agitator systemsoffering less shear forces than blades have been described. Agitationmay be driven either directly or indirectly by magnetically coupleddrives. Indirect drives reduce the risk of microbial contaminationthrough seals on stirrer shafts.

The airlift reactor, also initially described for microbial fermentationand later adapted for mammalian culture, relies on a gas stream to bothmix and oxygenate the culture. The gas stream enters a riser section ofthe reactor and drives circulation. Gas disengages at the culturesurface, causing denser liquid free of gas bubbles to travel downward inthe downcomer section of the reactor. The main advantage of this designis the simplicity and lack of need for mechanical mixing. Typically, theheight-to-diameter ratio is 10:1. The airlift reactor scales uprelatively easily, has good mass transfer of gasses and generatesrelatively low shear forces.

Most large-scale suspension cultures are operated as batch or fed-batchprocesses because they are the most straightforward to operate and scaleup. However, continuous processes based on chemostat or perfusionprinciples are available.

A batch process is a closed system in which a typical growth profile isseen. A lag phase is followed by exponential, stationary and declinephases. In such a system, the environment is continuously changing asnutrients are depleted and metabolites accumulate. This makes analysisof factors influencing cell growth and productivity, and henceoptimization of the process, a complex task. Productivity of a batchprocess may be increased by controlled feeding of key nutrients toprolong the growth cycle. Such a fed-batch process is still a closedsystem because cells, products and waste products are not removed.

In what is still a closed system, perfusion of fresh medium through theculture can be achieved by retaining the cells with a variety of devices(e.g. fine mesh spin filter, hollow fiber or flat plate membranefilters, settling tubes). Spin filter cultures can produce celldensities of approximately 5×10⁷ cells/ml. A true open system and thesimplest perfusion process is the chemostat in which there is an inflowof medium and an outflow of cells and products. Culture medium is fed tothe reactor at a predetermined and constant rate which maintains thedilution rate of the culture at a value less than the maximum specificgrowth rate of the cells (to prevent washout of the cell mass from thereactor). Culture fluid containing cells and cell products andbyproducts is removed at the same rate.

C) Non-Perfused Attachment Systems.

Traditionally, anchorage-dependent cell cultures are propagated on thebottom of small glass or plastic vessels. The restrictedsurface-to-volume ratio offered by classical and traditional techniques,suitable for the laboratory scale, has created a bottleneck in theproduction of cells and cell products on a large scale. In an attempt toprovide systems that offer large accessible surfaces for cell growth insmall culture volume, a number of techniques have been proposed: theroller bottle system, the stack plates propagator, the spiral filmbottles, the hollow fiber system, the packed bed, the plate exchangersystem, and the membrane tubing reel. Since these systems arenon-homogeneous in their nature, and are sometimes based on multipleprocesses, they suffer from the following shortcomings—limited potentialfor scale-up, difficulties in taking cell samples, limited potential formeasuring and controlling key process parameters and difficulty inmaintaining homogeneous environmental conditions throughout the culture.

Despite these drawbacks, a commonly used process for large scaleanchorage-dependent cell production is the roller bottle. Being littlemore than a large, differently shaped T-flask, simplicity of the systemmakes it very dependable and, hence, attractive. Fully automated robotsare available that can handle thousands of roller bottles per day, thuseliminating the risk of contamination and inconsistency associated withthe otherwise required intense human handling. With frequent mediachanges, roller bottle cultures can achieve cell densities of close to0.5×10⁶ cells/cm² (corresponding to approximately 10⁹ cells/bottle oralmost 10⁷ cells/ml of culture media).

D) Cultures on Microcarriers

In an effort to overcome the shortcomings of the traditionalanchorage-dependent culture processes, van Wezel (1967) developed theconcept of the microcarrier culturing systems. In this system, cells arepropagated on the surface of small solid particles suspended in thegrowth medium by slow agitation. Cells attach to the microcarriers andgrow gradually to confluency on the microcarrier surface. In fact, thislarge scale culture system upgrades the attachment dependent culturefrom a single disc process to a unit process in which both monolayer andsuspension culture have been brought together. Thus, combining thenecessary surface for a cell to grow with the advantages of thehomogeneous suspension culture increases production.

The advantages of microcarrier cultures over most otheranchorage-dependent, large-scale cultivation methods are several fold.First, microcarrier cultures offer a high surface-to-volume ratio(variable by changing the carrier concentration) which leads to highcell density yields and a potential for obtaining highly concentratedcell products. Cell yields are up to 1-2×10⁷ cells/ml when cultures arepropagated in a perfused reactor mode. Second, cells can be propagatedin one unit process vessels instead of using many small low-productivityvessels (i.e., flasks or dishes). This results in far better nutrientutilization and a considerable saving of culture medium. Moreover,propagation in a single reactor leads to reduction in need for facilityspace and in the number of handling steps required per cell, thusreducing labor cost and risk of contamination. Third, the well-mixed andhomogeneous microcarrier suspension culture makes it possible to monitorand control environmental conditions (e.g., pH, pO₂, and concentrationof medium components), thus leading to more reproducible cellpropagation and product recovery. Fourth, it is possible to take arepresentative sample for microscopic observation, chemical testing, orenumeration. Fifth, since microcarriers settle out of suspensionquickly, use of a fed-batch process or harvesting of cells can be donerelatively easily. Sixth, the mode of the anchorage-dependent culturepropagation on the microcarriers makes it possible to use this systemfor other cellular manipulations, such as cell transfer without the useof proteolytic enzymes, cocultivation of cells, transplantation intoanimals, and perfusion of the culture using decanters, columns,fluidized beds, or hollow fibers for microcarrier retainment. Seventh,microcarrier cultures are relatively easily scaled up using conventionalequipment used for cultivation of microbial and animal cells insuspension.

E) Microencapsulation of Mammalian Cells

One method which has shown to be particularly useful for culturingmammalian cells is microencapsulation. The mammalian cells are retainedinside a semipermeable hydrogel membrane. A porous membrane is formedaround the cells permitting the exchange of nutrients, gases, andmetabolic products with the bulk medium surrounding the capsule. Severalmethods have been developed that are gentle, rapid and non-toxic andwhere the resulting membrane is sufficiently porous and strong tosustain the growing cell mass throughout the term of the culture. Thesemethods are all based on soluble alginate gelled by droplet contact witha calcium-containing solution. Lim (1982, U.S. Pat. No. 4,352,883,incorporated herein by reference,) describes cells concentrated in anapproximately 1% solution of sodium alginate which are forced through asmall orifice, forming droplets, and breaking free into an approximately1% calcium chloride solution. The droplets are then cast in a layer ofpolyamino acid that ionically bonds to the surface alginate. Finally thealginate is reliquefied by treating the droplet in a chelating agent toremove the calcium ions. Other methods use cells in a calcium solutionto be dropped into a alginate solution, thus creating a hollow alginatesphere. A similar approach involves cells in a chitosan solution droppedinto alginate, also creating hollow spheres.

Microencapsulated cells are easily propagated in stirred tank reactorsand, with beads sizes in the range of 150-1500 μm in diameter, areeasily retained in a perfused reactor using a fine-meshed screen. Theratio of capsule volume to total media volume can be maintained from asdense as 1:2 to 1:10. With intracapsular cell densities of up to 108,the effective cell density in the culture is 1-5×10⁷.

The advantages of microencapsulation over other processes include theprotection from the deleterious effects of shear stresses which occurfrom sparging and agitation, the ability to easily retain beads for thepurpose of using perfused systems, scale up is relativelystraightforward and the ability to use the beads for implantation.

The current invention includes cells which are anchorage-dependent innature. 293 cells, for example, are anchorage-dependent, and when grownin suspension, the cells will attach to each other and grow in clumps,eventually suffocating cells in the inner core of each clump as theyreach a size that leaves the core cells unsustainable by the cultureconditions. Therefore, an efficient means of large-scale culture ofanchorage-dependent cells is needed in order to effectively employ thesecells to generate large quantities of adenovirus.

F) Perfused Attachment Systems

Perfused attachment systems are a preferred form of the presentinvention. Perfusion refers to continuous flow at a steady rate, throughor over a population of cells (of a physiological nutrient solution). Itimplies the retention of the cells within the culture unit as opposed tocontinuous-flow culture which washes the cells out with the withdrawnmedia (e.g., chemostat). The idea of perfusion has been known since thebeginning of the century, and has been applied to keep small pieces oftissue viable for extended microscopic observation. The technique wasinitiated to mimic the cells milieu in vivo where cells are continuouslysupplied with blood, lymph, or other body fluids. Without perfusion,cells in culture go through alternating phases of being fed and starved,thus limiting full expression of their growth and metabolic potential.

The current use of perfused culture is in response to the challenge ofgrowing cells at high densities (i.e., 0.1-5×10⁸ cells/ml). In order toincrease densities beyond 2-4×10⁶ cells/ml, the medium has to beconstantly replaced with a fresh supply in order to make up fornutritional deficiencies and to remove toxic products. Perfusion allowsfor a far better control of the culture environment (pH, pO₂, nutrientlevels, etc.) and is a means of significantly increasing the utilizationof the surface area within a culture for cell attachment.

The development of a perfused packed-bed reactor using a bed matrix of anon-woven fabric has provided a means for maintaining a perfusionculture at densities exceeding 10⁸ cells/ml of the bed volume(CelliGen™, New Brunswick Scientific, Edison, N.J.; Wang et al., 1992;Wang et al., 1993; Wang et al., 1994). Briefly described, this reactorcomprises an improved reactor for culturing of both anchorage- andnon-anchorage-dependent cells. The reactor is designed as a packed bedwith a means to provide internal recirculation. Preferably, a fibermatrix carrier is placed in a basket within the reactor vessel. A topand bottom portion of the basket has holes, allowing the medium to flowthrough the basket. A specially designed impeller provides recirculationof the medium through the space occupied by the fiber matrix forassuring a uniform supply of nutrient and the removal of wastes. Thissimultaneously assures that a negligible amount of the total cell massis suspended in the medium. The combination of the basket and therecirculation also provides a bubble-free flow of oxygenated mediumthrough the fiber matrix. The fiber matrix is a non-woven fabric havinga “pore” diameter of from 10 μm to 100 μm, providing for a high internalvolume with pore volumes corresponding to 1 to 20 times the volumes ofindividual cells.

In comparison to other culturing systems, this approach offers severalsignificant advantages. With a fiber matrix carrier, the cells areprotected against mechanical stress from agitation and foaming. The freemedium flow through the basket provides the cells with optimum regulatedlevels of oxygen, pH, and nutrients. Products can be continuouslyremoved from the culture and the harvested products are free of cellsand can be produced in low-protein medium which facilitates subsequentpurification steps. Also, the unique design of this reactor systemoffers an easier way to scale up the reactor. Currently, sizes up to 30liter are available. One hundred liter and 300 liter versions are indevelopment and theoretical calculations support up to a 1000 literreactor. This technology is explained in detail in WO 94/17178 (Aug. 4,1994, Freedman et al.), which is hereby incorporated by reference in itsentirety.

The Cellcube™ (Corning-Costar) module provides a large styrenic surfacearea for the immobilization and growth of substrate attached cells. Itis an integrally encapsulated sterile single-use device that has aseries of parallel culture plate joined to create thin sealed laminarflow spaces between adjacent plates.

The Cellcube™ module has inlet and outlet ports that are diagonallyopposite each other and help regulate the flow of media. During thefirst few days of growth the culture is generally satisfied by the mediacontained within the system after initial seeding. The amount of timebetween the initial seeding and the start of the media perfusion isdependent on the density of cells in the seeding inoculum and the cellgrowth rate. The measurement of nutrient concentration in thecirculating media is a good indicator of the status of the culture. Whenestablishing a procedure it may be necessary to monitor the nutrientscomposition at a variety of different perfusion rates to determine themost economical and productive operating parameters.

Cells within the system reach a higher density of solution (cells/ml)than in traditional culture systems. Many typically used basal media aredesigned to support 1-2×10⁶ cells/ml/day. A typical Cellcube™, run withan 85,000 cm² surface, contains approximately 6 L media within themodule. The cell density often exceeds 10⁷ cells/mL in the culturevessel. At confluence, 2-4 reactor volumes of media are required perday.

The timing and parameters of the production phase of cultures depends onthe type and use of a particular cell line. Many cultures require adifferent media for production than is required for the growth phase ofthe culture. The transition from one phase to the other will likelyrequire multiple washing steps in traditional cultures. However, theCellcube™ system employs a perfusion system. On of the benefits of sucha system is the ability to provide a gentle transition between variousoperating phases. The perfusion system negates the need for traditionalwash steps that seek to remove serum components in a growth medium.

In an exemplary embodiment of the present invention, the CellCube™system is used to grow cells transfected with AdCMVp53. 293 cells wereinoculated into the Cellcube™ according to the manufacturer'srecommendation. Inoculation cell densities were in the range of1-1.5×10⁴/cm². Cells were allowed to grow for 7 days at 37° C. underculture conditions of pH=7.20, DO=60% air saturation. The mediumperfusion rate was regulated according to the glucose concentration inthe Cellcube™. One day before viral infection, medium for perfusion waschanged from a buffer comprising 10% FBS to a buffer comprising 2% FBS.On day 8, cells were infected with virus at a multiplicity of infection(MOI) of 5. Medium perfusion was stopped for 1 hr immediately afterinfection then resumed for the remaining period of the virus productionphase. Culture was harvested 45-48 hr post-infection. Of course theseculture conditions are exemplary and may be varied according to thenutritional needs and growth requirements of a particular cell line.Such variation may be performed without undue experimentation and arewell within the skill of the ordinary person in the art.

G) Serum-Free Suspension Culture

In particular embodiments, adenoviral vectors for gene therapy areproduced from anchorage-dependent culture of 293 cells (293A cells) asdescribed above. Scale-up of adenoviral vector production is constrainedby the anchorage-dependency of 293A cells. To facilitate scale-up andmeet future demand for adenoviral vectors, significant efforts have beendevoted to the development of alternative production processes that areamenable to scale-up. Methods include growing 293A cells in microcarriercultures and adaptation of 293A producer cells into suspension cultures.Microcarrier culture techniques have been described above. Thistechnique relies on the attachment of producer cells onto the surfacesof microcarriers which are suspended in culture media by mechanicalagitation. The requirement of cell attachment may present somelimitations to the scaleability of microcarrier cultures.

Until the present application there have been no reports on the use of293 suspension cells for adenoviral vector production for gene therapy.Furthermore, the reported suspension 293 cells require the presence of5-10% FBS in the culture media for optimal cell growth and virusproduction. Historically, presence of bovine source proteins in cellculture media has been a regulatory concerns, especially recentlybecause of the outbreak of Bovine Spongiform Encephalopathy (BSE) insome countries. Rigorous and complex downstream purification process hasto be developed to remove contaminating proteins and any adventitiousviruses from the final product. Development of serum-free 293 suspensionculture is deemed to be a major process improvement for the productionof adenoviral vector for gene therapy.

Results of virus production in spinner flasks and a 3 L stirred tankbioreactor indicate that cell specific virus productivity of the 293SFcells was approximately 2.5×10⁴ vp/cell, which is approximately 60-90%of that from the 293A cells. However, because of the higher stationarycell concentration, volumetric virus productivity from the 293SF cultureis essentially equivalent to that of the 293A cell culture. Theinventors also observed that virus production increased significantly bycarrying out a fresh medium exchange at the time of virus infection. Theinventors are going to evaluate the limiting factors in the medium.

These findings allow for a scaleable, efficient, and easily validatableprocess for the production adenoviral vector. This adaptation method isnot limited to 293A cells only and will be equally useful when appliedto other adenoviral vector producer cells.

3. METHODS OF CELL HARVEST AND LYSIS

Adenoviral infection results in the lysis of the cells being infected.The lytic characteristics of adenovirus infection permit two differentmodes of virus production. One is harvesting infected cells prior tocell lysis. The other mode is harvesting virus supernatant aftercomplete cell lysis by the produced virus. For the latter mode, longerincubation times are required in order to achieve complete cell lysis.This prolonged incubation time after virus infection creates a seriousconcern about increased possibility of generation of replicationcompetent adenovirus (RCA), particularly for the current firstgeneration adenoviral vectors (E1-deleted vector). Therefore, harvestinginfected cells before cell lysis was chosen as the production mode ofchoice. Table 1 lists the most common methods that have been used forlysing cells after cell harvest. TABLE 1 Methods used for cell lysisMethods Procedures Comments Freeze-thaw Cycling between dry ice and Easyto carry out at lab 37° C. water bath scale. High cell lysis efficiencyNot scaleable Not recommended for large scale manufacturing Solid ShearFrench Press Capital equipment Hughes Press investment Virus containmentconcerns Lack of experience Detergent lysis Non-ionic detergent Easy tocarry out at both lab solutions such as Tween, and manufacturing Triton,NP-40, etc. scale Wide variety of detergent choices Concerns of residualdetergent in finished product Hypotonic solution lysis water, citricbuffer Low lysis efficiency Liquid Shear Homogenizer Capital equipmentImpinging Jet investment Microfluidizer Virus containment concernsScaleability concerns Sonication ultrasound Capital equipment investmentVirus containment concerns Noise pollution Scaleability concern

A) Detergents

Cells are bounded by membranes. In order to release components of thecell, it is necessary to break open the cells. The most advantageous wayin which this can be accomplished, according to the present invention,is to solubilize the membranes with the use of detergents. Detergentsare amphipathic molecules with an apolar end of aliphatic or aromaticnature and a polar end which may be charged or uncharged. Detergents aremore hydrophilic than lipids and thus have greater water solubility thanlipids. They allow for the dispersion of water insoluble compounds intoaqueous media and are used to isolate and purify proteins in a nativeform.

Detergents can be denaturing or non-denaturing. The former can beanionic such as sodium dodecyl sulfate or cationic such as ethyltrimethyl ammonium bromide. These detergents totally disrupt membranesand denature the protein by breaking protein-protein interactions. Nondenaturing detergents can be divided into non-anionic detergents such asTriton®X-100, bile salts such as cholates and zwitterionic detergentssuch as CHAPS. Zwitterionics contain both cationic and anion groups inthe same molecule, the positive electric charge is neutralized by thenegative charge on the same or adjacent molecule.

Denaturing agents such as SDS bind to proteins as monomers and thereaction is equilibrium driven until saturated. Thus, the freeconcentration of monomers determines the necessary detergentconcentration. SDS binding is cooperative i.e. the binding of onemolecule of SDS increase the probability of another molecule binding tothat protein, and alters proteins into rods whose length is proportionalto their molecular weight.

Non-denaturing agents such as Triton®X-100 do not bind to nativeconformations nor do they have a cooperative binding mechanism. Thesedetergents have rigid and bulky apolar moieties that do not penetrateinto water soluble proteins. They bind to the hydrophobic parts ofproteins. Triton®X100 and other polyoxyethylene nonanionic detergentsare inefficient in breaking protein-protein interaction and can causeartifactual aggregations of protein. These detergents will, however,disrupt protein-lipid interactions but are much gentler and capable ofmaintaining the native form and functional capabilities of the proteins.

Detergent removal can be attempted in a number of ways. Dialysis workswell with detergents that exist as monomers. Dialysis is somewhatineffective with detergents that readily aggregate to form micellesbecause they micelles are too large to pass through dialysis. Ionexchange chromatography can be utilized to circumvent this problem. Thedisrupted protein solution is applied to an ion exchange chromatographycolumn and the column is then washed with buffer minus detergent. Thedetergent will be removed as a result of the equilibration of the bufferwith the detergent solution. Alternatively the protein solution may bepassed through a density gradient. As the protein sediments through thegradients the detergent will come off due to the chemical potential.

Often a single detergent is not versatile enough for the solubilizationand analysis of the milieu of proteins found in a cell. The proteins canbe solubilized in one detergent and then placed in another suitabledetergent for protein analysis. The protein detergent micelles formed inthe first step should separate from pure detergent micelles. When theseare added to an excess of the detergent for analysis, the protein isfound in micelles with both detergents. Separation of thedetergent-protein micelles can be accomplished with ion exchange or gelfiltration chromatography, dialysis or buoyant density type separations.

Triton®X-Detergents: This family of detergents (Triton®X-100, X114 andNP-40) have the same basic characteristics but are different in theirspecific hydrophobic-hydrophilic nature. All of these heterogeneousdetergents have a branched 8-carbon chain attached to an aromatic ring.This portion of the molecule contributes most of the hydrophobic natureof the detergent. Triton®X detergents are used to solublize membraneproteins under non-denaturing conditions. The choice of detergent tosolubilize proteins will depend on the hydrophobic nature of the proteinto be solubilized. Hydrophobic proteins require hydrophobic detergentsto effectively solubilize them.

Triton®X-100 and NP-40 are very similar in structure and hydrophobicityand are interchangeable in most applications including cell lysis,delipidation protein dissociation and membrane protein and lipidsolubilization. Generally 2 mg detergent is used to solubilize 1 mgmembrane protein or 10 mg detergent/1 mg of lipid membrane. Triton®X-114is useful for separating hydrophobic from hydrophilic proteins.

Brij® Detergents: These are similar in structure to Triton®X detergentsin that they have varying lengths of polyoxyethylene chains attached toa hydrophobic chain. However, unlike Triton®X detergents, the Brij®detergents do not have an aromatic ring and the length of the carbonchains can vary. The Brij® detergents are difficult to remove fromsolution using dialysis but may be removed by detergent removing gels.Brij®58 is most similar to Triton®X100 in its hydrophobic/hydrophiliccharacteristics. Brij®-35 is a commonly used detergent in HPLCapplications.

Dializable Nonionic Detergents: η-Octyl-β-D-glucoside(octylglucopyranoside) and η-Octyl-β-D-thioglucoside(octylthioglucopyranoside, OTG) are nondenaturing nonionic detergentswhich are easily dialyzed from solution. These detergents are useful forsolubilizing membrane proteins and have low UV absorbances at 280 nm.Octylglucoside has a high CMC of 23-25 mM and has been used atconcentrations of 1.1-1.2% to solubilize membrane proteins.

Octylthioglucoside was first synthesized to offer an alternative tooctylglucoside. Octylglucoside is expensive to manufacture and there aresome inherent problems in biological systems because it can behydrolyzed by α-glucosidase.

Tween® Detergents: The Tween® detergents are nondenaturing, nonionicdetergents. They are polyoxyethylene sorbitan esters of fatty acids.Tween® 20 and Tween® 80 detergents are used as blocking agents inbiochemical applications and are usually added to protein solutions toprevent nonspecific binding to hydrophobic materials such as plastics ornitrocellulose. They have been used as blocking agents in ELISA andblotting applications. Generally, these detergents are used atconcentrations of 0.01-1.0% to prevent nonspecific binding tohydrophobic materials.

Tween® 20 and other nonionic detergents have been shown to remove someproteins from the surface of nitrocellulose. Tween® 80 has been used tosolubilize membrane proteins, present nonspecific binding of protein tomultiwell plastic tissue culture plates and to reduce nonspecificbinding by serum proteins and biotinylated protein A to polystyreneplates in ELISA.

The difference between these detergents is the length of the fatty acidchain. Tween® 80 is derived from oleic acid with a C₁₈ chain whileTween® 20 is derived from lauric acid with a C₁₂ chain. The longer fattyacid chain makes the Tween® 80 detergent less hydrophilic than Tween® 20detergent. Both detergents are very soluble in water.

The Tween® detergents are difficult to remove from solution by dialysis,but Tween® 20 can be removed by detergent removing gels. Thepolyoxyethylene chain found in these detergents makes them subject tooxidation (peroxide formation) as is true with the Triton® X and Brij®series detergents.

Zwitterionic Detergents: The zwitterionic detergent, CHAPS, is asulfobetaine derivative of cholic acid. This zwitterionic detergent isuseful for membrane protein solubilization when protein activity isimportant. This detergent is useful over a wide range of pH (pH 2-12)and is easily removed from solution by dialysis due to high CMCs (8-10mM). This detergent has low absorbances at 280 nm making it useful whenprotein monitoring at this wavelength is necessary. CHAPS is compatiblewith the BCA Protein Assay and can be removed from solution by detergentremoving gel. Proteins can be iodinated in the presence of CHAPS

CHAPS has been successfully used to solubilize intrinsic membraneproteins and receptors and maintain the functional capability of theprotein. When cytochrome P-450 is solubilized in either Triton® X-100 orsodium cholate aggregates are formed.

B) Non-Detergent Methods

Various non-detergent methods, though not preferred, may be employed inconjunction with other advantageous aspects of the present invention:

Freeze-Thaw: This has been a widely used technique for lysis cells in agentle and effective manner. Cells are generally frozen rapidly in, forexample, a dry ice/ethanol bath until completely frozen, thentransferred to a 37° C. bath until completely thawed. This cycle isrepeated a number of times to achieve complete cell lysis.

Sonication: High frequency ultrasonic oscillations have been found to beuseful for cell disruption. The method by which ultrasonic waves breakcells is not fully understood but it is known that high transientpressures are produced when suspensions are subjected to ultrasonicvibration. The main disadvantage with this technique is thatconsiderable amounts of heat are generated. In order to minimize heateffects specifically designed glass vessels are used to hold the cellsuspension. Such designs allow the suspension to circulate away from theultrasonic probe to the outside of the vessel where it is cooled as theflask is suspended in ice.

High Pressure Extrusion: This is a frequently used method to disruptmicrobial cell. The French pressure cell employs pressures of 10.4×10⁷Pa (16, 000 p.s.i) to break cells open. These apparatus consists of astainless steel chamber which opens to the outside by means of a needlevalve. The cell suspension is placed in the chamber with the needlevalve in the closed position. After inverting the chamber, the valve isopened and the piston pushed in to force out any air in the chamber.With the valve in the closed position, the chamber is restored to itsoriginal position, placed on a solid based and the required pressure isexerted on the piston by a hydraulic press. When the pressure has beenattained the needle valve is opened fractionally to slightly release thepressure and as the cells expand they burst. The valve is kept openwhile the pressure is maintained so that there is a trickle of rupturedcell which may be collected.

Solid Shear Methods: Mechanical shearing with abrasives may be achievedin Mickle shakers which oscillate suspension vigorously (300-3000time/min) in the presence of glass beads of 500 nm diameter. This methodmay result in organelle damage. A more controlled method is to use aHughes press where a piston forces most cells together with abrasives ordeep frozen paste of cells through a 0.25 mm diameter slot in thepressure chamber. Pressures of up to 5.5×10⁷ Pa (8000 p.s.i.) may beused to lyse bacterial preparations.

Liquid Shear Methods: These methods employ blenders, which use highspeed reciprocating or rotating blades, homogenizers which use anupward/downward motion of a plunger and ball and microfluidizers orimpinging jets which use high velocity passage through small diametertubes or high velocity impingement of two fluid streams. The blades ofblenders are inclined at different angles to permit efficient mixing.Homogenizers are usually operated in short high speed bursts of a fewseconds to minimize local heat. These techniques are not generallysuitable for microbial cells but even very gentle liquid shear isusually adequate to disrupt animal cells.

Hypotonic/Hypertonic Methods: Cells are exposed to a solution with amuch lower (hypotonic) or higher (hypertonic) solute concentration. Thedifference in solute concentration creates an osmotic pressure gradient.The resulting flow of water into the cell in a hypotonic environmentcauses the cells to swell and burst. The flow of water out of the cellin a hypertonic environment causes the cells to shrink and subsequentlyburst.

4. METHODS OF CONCENTRATION AND FILTRATION

One aspect of the present invention employs methods of crudepurification of adenovirus from a cell lysate. These methods includeclarification, concentration and diafiltration. The initial step in thispurification process is clarification of the cell lysate to remove largeparticulate matter, particularly cellular components, from the celllysate. Clarification of the lysate can be achieved using a depth filteror by tangential flow filtration. In a preferred embodiment of thepresent invention, the cell lysate is passed through a depth filter,which consists of a packed column of relatively non-adsorbent material(e.g. polyester resins, sand, diatomeceous earth, colloids, gels, andthe like). In tangential flow filtration (TFF), the lysate solutionflows across a membrane surface which facilitates back diffusion ofsolute from the membrane surface into the bulk solution. Membranes aregenerally arranged within various types of filter apparatus includingopen channel plate and frame, hollow fibers, and tubules.

After clarification and prefiltration of the cell lysate, the resultantvirus supernatant is first concentrated and then the buffer is exchangedby diafiltration. The virus supernatant is concentrated by tangentialflow filtration across an ultrafiltration membrane of 100-300K nominalmolecular weight cutoff. Ultrafiltration is a pressure-modifiedconvective process that uses semi-permeable membranes to separatespecies by molecular size, shape and/or charge. It separates solventsfrom solutes of various sizes, independent of solute molecular size.Ultrafiltration is gentle, efficient and can be used to simultaneouslyconcentrate and desalt solutions. Ultrafiltration membranes generallyhave two distinct layers: a thin (0.1-1.5 μm), dense skin with a porediameter of 10-400 angstroms and an open substructure of progressivelylarger voids which are largely open to the permeate side of theultrafilter. Any species capable of passing through the pores of theskin can therefore freely pass through the membrane. For maximumretention of solute, a membrane is selected that has a nominal molecularweight cut-off well below that of the species being retained. Inmacromolecular concentration, the membrane enriches the content of thedesired biological species and provides filtrate cleared of retainedsubstances. Microsolutes are removed convectively with the solvent. Asconcentration of the retained solute increases, the ultrafiltration ratediminishes.

Diafiltration, or buffer exchange, using ultrafilters is an ideal wayfor removal and exchange of salts, sugars, non-aqueous solventsseparation of free from bound species, removal of material of lowmolecular weight, or rapid change of ionic and pH environments.Microsolutes are removed most efficiently by adding solvent to thesolution being ultrafiltered at a rate equal to the ultrafiltrationrate. This washes microspecies from the solution at constant volume,purifying the retained species. The present invention utilizes adiafiltration step to exchange the buffer of the virus supernatant priorto Benzonase® treatment.

5. VIRAL INFECTION

The present invention employs, in one example, adenoviral infection ofcells in order to generate therapeutically significant vectors.Typically, the virus will simply be exposed to the appropriate host cellunder physiologic conditions, permitting uptake of the virus. Thoughadenovirus is exemplified, the present methods may be advantageouslyemployed with other viral vectors, as discussed below.

A) Adenovirus

Adenovirus is particularly suitable for use as a gene transfer vectorbecause of its mid-sized DNA genome, ease of manipulation, high titer,wide target-cell range, and high infectivity. The roughly 36 kB viralgenome is bounded by 100-200 base pair (bp) inverted terminal repeats(ITR), in which are contained cis-acting elements necessary for viralDNA replication and packaging. The early (E) and late (L) regions of thegenome that contain different transcription units are divided by theonset of viral DNA replication.

The E1 region (E1A and E1B) encodes proteins responsible for theregulation of transcription of the viral genome and a few cellulargenes. The expression of the E2 region (E2A and E2B) results in thesynthesis of the proteins for viral DNA replication. These proteins areinvolved in DNA replication, late gene expression, and host cell shutoff (Renan, 1990). The products of the late genes (L1, L2, L3, L4 andL5), including the majority of the viral capsid proteins, are expressedonly after significant processing of a single primary transcript issuedby the major late promoter (MLP). The MLP (located at 16.8 map units) isparticularly efficient during the late phase of infection, and all themRNAs issued from this promoter possess a 5′ tripartite leader (TL)sequence which makes them preferred mRNAs for translation.

In order for adenovirus to be optimized for gene therapy, it isnecessary to maximize the carrying capacity so that large segments ofDNA can be included. It also is very desirable to reduce the toxicityand immunologic reaction associated with certain adenoviral products.Elimination of large potions of the adenoviral genome, and providing thedelete gene products in trans, by helper virus and/or helper cells,allows for the insertion of large portions of heterologous DNA into thevector. This strategy also will result in reduced toxicity andimmunogenicity of the adenovirus gene products.

The large displacement of DNA is possible because the cis elementsrequired for viral DNA replication all are localized in the invertedterminal repeats (ITR) (100-200 bp) at either end of the linear viralgenome. Plasmids containing ITR's can replicate in the presence of anon-defective adenovirus (Hay et al., 1984). Therefore, inclusion ofthese elements in an adenoviral vector should permit replication.

In addition, the packaging signal for viral encapsidation is localizedbetween 194-385 bp (0.5-1.1 map units) at the left end of the viralgenome (Hearing et al., 1987). This signal mimics the proteinrecognition site in bacteriophage λ DNA where a specific sequence closeto the left end, but outside the cohesive end sequence, mediates thebinding to proteins that are required for insertion of the DNA into thehead structure. E1 substitution vectors of Ad have demonstrated that a450 bp (0-1.25 map units) fragment at the left end of the viral genomecould direct packaging in 293 cells (Levrero et al., 1991).

Previously, it has been shown that certain regions of the adenoviralgenome can be incorporated into the genome of mammalian cells and thegenes encoded thereby expressed. These cell lines are capable ofsupporting the replication of an adenoviral vector that is deficient inthe adenoviral function encoded by the cell line. There also have beenreports of complementation of replication deficient adenoviral vectorsby “helping” vectors, e.g., wild-type virus or conditionally defectivemutants.

Replication-deficient adenoviral vectors can be complemented, in trans,by helper virus. This observation alone does not permit isolation of thereplication-deficient vectors, however, since the presence of helpervirus, needed to provide replicative functions, would contaminate anypreparation. Thus, an additional element was needed that would addspecificity to the replication and/or packaging of thereplication-deficient vector. That element, as provided for in thepresent invention, derives from the packaging function of adenovirus.

It has been shown that a packaging signal for adenovirus exists in theleft end of the conventional adenovirus map (Tibbetts, 1977). Laterstudies showed that a mutant with a deletion in the E1A (194-358 bp)region of the genome grew poorly even in a cell line that complementedthe early (E1A) function (Hearing and Shenk, 1983). When a compensatingadenoviral DNA (0-353 bp) was recombined into the right end of themutant, the virus was packaged normally. Further mutational analysisidentified a short, repeated, position-dependent element in the left endof the Ad5 genome. One copy of the repeat was found to be sufficient forefficient packaging if present at either end of the genome, but not whenmoved towards the interior of the Ad5 DNA molecule (Hearing et al.,1987).

By using mutated versions of the packaging signal, it is possible tocreate helper viruses that are packaged with varying efficiencies.Typically, the mutations are point mutations or deletions. When helperviruses with low efficiency packaging are grown in helper cells, thevirus is packaged, albeit at reduced rates compared to wild-type virus,thereby permitting propagation of the helper. When these helper virusesare grown in cells along with virus that contains wild-type packagingsignals, however, the wild-type packaging signals are recognizedpreferentially over the mutated versions. Given a limiting amount ofpackaging factor, the virus containing the wild-type signals arepackaged selectively when compared to the helpers. If the preference isgreat enough, stocks approaching homogeneity should be achieved.

B) Retrovirus

Although adenoviral infection of cells for the generation oftherapeutically significant vectors is a preferred embodiments of thepresent invention, it is contemplated that the present invention mayemploy retroviral infection of cells for the purposes of generating suchvectors. The retroviruses are a group of single-stranded RNA virusescharacterized by an ability to convert their RNA to double-stranded DNAin infected cells by a process of reverse-transcription (Coffin, 1990).The resulting DNA then stably integrates into cellular chromosomes as aprovirus and directs synthesis of viral proteins. The integrationresults in the retention of the viral gene sequences in the recipientcell and its descendants. The retroviral genome contains threegenes—gag, pol and env—that code for capsid proteins, polymerase enzyme,and envelope components, respectively. A sequence found upstream fromthe gag gene, termed Y, functions as a signal for packaging of thegenome into virions. Two long terminal repeat (LTR) sequences arepresent at the 5′ and 3′ ends of the viral genome. These contain strongpromoter and enhancer sequences and are also required for integration inthe host cell genome (Coffin, 1990).

In order to construct a retroviral vector, a nucleic acid encoding apromoter is inserted into the viral genome in the place of certain viralsequences to produce a virus that is replication-defective. In order toproduce virions, a packaging cell line containing the gag, pol and envgenes but without the LTR and Y components is constructed (Mann et al.,1983). When a recombinant plasmid containing a human cDNA, together withthe retroviral LTR and Y sequences is introduced into this cell line (bycalcium phosphate precipitation for example), the Y sequence allows theRNA transcript of the recombinant plasmid to be packaged into viralparticles, which are then secreted into the culture media (Nicolas andRubenstein, 1988; Temin, 1986; Mann et al., 1983). The media containingthe recombinant retroviruses is then collected, optionally concentrated,and used for gene transfer. Retroviral vectors are able to infect abroad variety of cell types. However, integration and stable expressionrequire the division of host cells (Paskind et al., 1975).

A novel approach designed to allow specific targeting of retrovirusvectors was recently developed based on the chemical modification of aretrovirus by the chemical addition of galactose residues to the viralenvelope. This modification could permit the specific infection of cellssuch as hepatocytes via asialoglycoprotein receptors, should this bedesired.

A different approach to targeting of recombinant retroviruses wasdesigned in which biotinylated antibodies against a retroviral envelopeprotein and against a specific cell receptor were used. The antibodieswere coupled via the biotin components by using streptavidin (Roux etal., 1989). Using antibodies against major histocompatibility complexclass I and class II antigens, the infection of a variety of human cellsthat bore those surface antigens was demonstrated with an ecotropicvirus in vitro (Roux et al., 1989).

C) Other Viral Vectors

Other viral vectors may be employed as expression constructs in thepresent invention. Vectors derived from viruses such as vaccinia virus(Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988),adeno-associated virus (AAV) (Ridgeway, 1988; Baichwal and Sugden, 1986;Hermonat and Muzycska, 1984) and herpesviruses may be employed. Theseviruses offer several features for use in gene transfer into variousmammalian cells.

6. ENGINEERING OF VIRAL VECTORS

In certain embodiments, the present invention further involves themanipulation of viral vectors. Such methods involve the use of a vectorconstruct containing, for example, a heterologous DNA encoding a gene ofinterest and a means for its expression, replicating the vector in anappropriate helper cell, obtaining viral particles produced therefrom,and infecting cells with the recombinant virus particles. The gene couldsimply encode a protein for which large quantities of the protein aredesired, i.e., large scale in vitro production methods. Alternatively,the gene could be a therapeutic gene, for example to treat cancer cells,to express immunomodulatory genes to fight viral infections, or toreplace a gene's function as a result of a genetic defect. In thecontext of the gene therapy vector, the gene will be a heterologous DNA,meant to include DNA derived from a source other than the viral genomewhich provides the backbone of the vector. Finally, the virus may act asa live viral vaccine and express an antigen of interest for theproduction of antibodies thereagainst. The gene may be derived from aprokaryotic or eukaryotic source such as a bacterium, a virus, a yeast,a parasite, a plant, or even an animal. The heterologous DNA also may bederived from more than one source, i.e., a multigene construct or afusion protein. The heterologous DNA may also include a regulatorysequence which may be derived from one source and the gene from adifferent source.

A) Therapeutic Genes

p53 currently is recognized as a tumor suppressor gene (Montenarh,1992). High levels of mutant p53 have been found in many cellstransformed by chemical carcinogenesis, ultraviolet radiation, andseveral viruses, including SV40. The p53 gene is a frequent target ofmutational inactivation in a wide variety of human tumors and is alreadydocumented to be the most frequently-mutated gene in common humancancers (Mercer, 1992). It is mutated in over 50% of human NSCLC(Hollestein et al., 1991) and in a wide spectrum of other tumors.

The p53 gene encodes a 393-amino-acid phosphoprotein that can formcomplexes with host proteins such as large-T antigen and EIB. Theprotein is found in normal tissues and cells, but at concentrationswhich are generally minute by comparison with transformed cells or tumortissue. Interestingly, wild-type p53 appears to be important inregulating cell growth and division. Overexpression of wild-type p53 hasbeen shown in some cases to be anti-proliferative in human tumor celllines. Thus, p53 can act as a negative regulator of cell growth(Weinberg, 1991) and may directly suppress uncontrolled cell growth ordirectly or indirectly activate genes that suppress this growth. Thus,absence or inactivation of wild-type p53 may contribute totransformation. However, some studies indicate that the presence ofmutant p53 may be necessary for full expression of the transformingpotential of the gene.

Wild-type p53 is recognized as an important growth regulator in manycell types. Missense mutations are common for the p53 gene and are knownto occur in at least 30 distinct codons, often creating dominant allelesthat produce shifts in cell phenotype without a reduction tohomozygosity. Additionally, many of these dominant negative allelesappear to be tolerated in the organism and passed on in the germ line.Various mutant alleles appear to range from minimally dysfunctional tostrongly penetrant, dominant negative alleles (Weinberg, 1991).

Casey and colleagues have reported that transfection of DNA encodingwild-type p53 into two human breast cancer cell lines restores growthsuppression control in such cells (Casey et al., 1991). A similar effecthas also been demonstrated on transfection of wild-type, but not mutant,p53 into human lung cancer cell lines (Takahasi et al., 1992). p53appears dominant over the mutant gene and will select againstproliferation when transfected into cells with the mutant gene. Normalexpression of the transfected p53 is not detrimental to normal cellswith endogenous wild-type p53. Thus, such constructs might be taken upby normal cells without adverse effects. It is thus proposed that thetreatment of p53-associated cancers with wild-type p53 expressionconstructs will reduce the number of malignant cells or their growthrate. Furthermore, recent studies suggest that some p53 wild-type tumorsare 20 also sensitive to the effects of exogenous p53 expression.

The major transitions of the eukaryotic cell cycle are triggered bycyclin-dependent kinases, or CDK's. One CDK, cyclin-dependent kinase 4(CDK4), regulates progression through the G₁ phase. The activity of thisenzyme may be to phosphorylate Rb at late G₁. The activity of CDK4 iscontrolled by an activating subunit, D-type cyclin, and by an inhibitorysubunit, e.g. p16^(INK4), which has been biochemically characterized asa protein that specifically binds to and inhibits CDK4, and thus mayregulate Rb phosphorylation (Serrano et al., 1993; Serrano et al.,1995). Since the p16^(INK4) protein is a CDK4 inhibitor (Serrano, 1993),deletion of this gene may increase the activity of CDK4, resulting inhyperphosphorylation of the Rb protein. p16 also is known to regulatethe function of CDK6.

p16^(INK4) belongs to a newly described class of CDK-inhibitory proteinsthat also includes p16^(B), p21^(WAF1, CIP1, SDI1), and p27. The p16gene maps to 9p21, a chromosome region frequently deleted in many tumortypes. Homozygous deletions and mutations of the p16^(INK4) gene arefrequent in human tumor cell lines. This evidence suggests that thep16^(INK4) gene is a tumor suppressor gene. This interpretation has beenchallenged, however, by the observation that the frequency of thep16^(INK4) gene alterations is much lower in primary uncultured tumorsthan in cultured cell lines (Caldas et al., 1994; Cheng et al., 1994;Hussussian et al., 1994; Kamb et al., 1994a; Kamb et al., 1994b; Mori etal., 1994; Okamoto et al., 1994; Nobori et al., 1995; Orlow et al.,1994; Arap et al., 1995). Restoration of wild-type p16^(INK4) functionby transfection with a plasmid expression vector reduced colonyformation by some human cancer cell lines (Okamoto, 1994; Arap, 1995).

C-CAM is expressed in virtually all epithelial cells (Odin and Obrink,1987). C-CAM, with an apparent molecular weight of 105 kD, wasoriginally isolated from the plasma membrane of the rat hepatocyte byits reaction with specific antibodies that neutralize cell aggregation(Obrink, 1991). Recent studies indicate that, structurally, C-CAMbelongs to the immunoglobulin (Ig) superfamily and its sequence ishighly homologous to carcinoembryonic antigen (CEA) (Lin and Guidotti,1989). Using a baculovirus expression system, Cheung et al. (1993a;1993b and 1993c) demonstrated that the first Ig domain of C-CAM iscritical for cell adhesion activity.

Cell adhesion molecules, or CAMs are known to be involved in a complexnetwork of molecular interactions that regulate organ development andcell differentiation (Edelman, 1985). Recent data indicate that aberrantexpression of CAMs may be involved in the tumorigenesis of severalneoplasms; for example, decreased expression of E-cadherin, which ispredominantly expressed in epithelial cells, is associated with theprogression of several kinds of neoplasms (Edelman and Crossin, 1991;Frixen et al., 1991; Bussemakers et al., 1992; Matsura et al., 1992;Umbas et al., 1992). Also, Giancotti and Ruoslahti (1990) demonstratedthat increasing expression of α₅β₁ integrin by gene transfer can reducetumorigenicity of Chinese hamster ovary cells in vivo. C-CAM now hasbeen shown to suppress tumor growth in vitro and in vivo.

Other tumor suppressors that may be employed according to the presentinvention include RB, APC, DCC, NF-1, NF-2, WT-1, MEN-I, MEN-II, zac1,p73, BRCA1, VHL, FCC, MMAC1, MCC, p16, p21, p57, C-CAM, p27 and BRCA2.Inducers of apoptosis, such as Bax, Bak, Bcl-X_(S), Bik, Bid, Harakiri,Ad E1B, Bad and ICE-CED3 proteases, similarly could find use accordingto the present invention.

Various enzyme genes are of interest according to the present invention.Such enzymes include cytosine deaminase, hypoxanthine-guaninephosphoribosyltransferase, galactose-1-phosphate uridyltransferase,phenylalanine hydroxylase, glucocerbrosidase, sphingomyelinase,α-L-iduronidase, glucose-6-phosphate dehydrogenase, HSV thymidine kinaseand human thymidine kinase.

Hormones are another group of gene that may be used in the vectorsdescribed herein. Included are growth hormone, prolactin, placentallactogen, luteinizing hormone, follicle-stimulating hormone, chorionicgonadotropin, thyroid-stimulating hormone, leptin, adrenocorticotropin(ACTH), angiotensin I and II, β-endorphin, β-melanocyte stimulatinghormone (P-MSH), cholecystokinin, endothelin I, galanin, gastricinhibitory peptide (GIP), glucagon, insulin, lipotropins, neurophysins,somatostatin, calcitonin, calcitonin gene related peptide (CGRP),β-calcitonin gene related peptide, hypercalcemia of malignancy factor(1-40), parathyroid hormone-related protein (107-139) (PTH-rP),parathyroid hormone-related protein (107-111) (PTH-rP), glucagon-likepeptide (GLP-1), pancreastatin, pancreatic peptide, peptide YY, PHM,secretin, vasoactive intestinal peptide (VIP), oxytocin, vasopressin(AVP), vasotocin, enkephalinamide, metorphinamide, alpha melanocytestimulating hormone (alpha-MSH), atrial natriuretic factor (5-28) (ANF),amylin, amyloid P component (SAP-1), corticotropin releasing hormone(CRH), growth hormone releasing factor (GHRH), luteinizinghormone-releasing hormone (LHRH), neuropeptide Y, substance K(neurokinin A), substance P and thyrotropin releasing hormone (TRH).

Other classes of genes that are contemplated to be inserted into thevectors of the present invention include interleukins and cytokines.Interleukin 1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9,IL-10, IL-11 IL-12, GM-CSF and G-CSF.

Examples of diseases for which the present viral vector would be usefulinclude, but are not limited to, adenosine deaminase deficiency, humanblood clotting factor IX deficiency in hemophilia B, and cysticfibrosis, which would involve the replacement of the cystic fibrosistransmembrane receptor gene. The vectors embodied in the presentinvention could also be used for treatment of hyperproliferativedisorders such as rheumatoid arthritis or restenosis by transfer ofgenes encoding angiogenesis inhibitors or cell cycle inhibitors.Transfer of prodrug activators such as the HSV-TK gene can be also beused in the treatment of hyperpoliferative disorders, including cancer.

B) Antisense Constructs

Oncogenes such as ras, myc, neu, raf erb, src, fms, jun, trk, ret, gsp,hst, bcl and abl also are suitable targets. However, for therapeuticbenefit, these oncogenes would be expressed as an antisense nucleicacid, so as to inhibit the expression of the oncogene. The term“antisense nucleic acid” is intended to refer to the oligonucleotidescomplementary to the base sequences of oncogene-encoding DNA and RNA.Antisense oligonucleotides, when introduced into a target cell,specifically bind to their target nucleic acid and interfere withtranscription, RNA processing, transport and/or translation. Targetingdouble-stranded (ds) DNA with oligonucleotide leads to triple-helixformation; targeting RNA will lead to double-helix formation.

Antisense constructs may be designed to bind to the promoter and othercontrol regions, exons, introns or even exon-intron boundaries of agene. Antisense RNA constructs, or DNA encoding such antisense RNAs, maybe employed to inhibit gene transcription or translation or both withina host cell, either in vitro or in vivo, such as within a host animal,including a human subject. Nucleic acid sequences comprising“complementary nucleotides” are those which are capable of base-pairingaccording to the standard Watson-Crick complementarity rules. That is,that the larger purines will base pair with the smaller pyrimidines toform only combinations of guanine paired with cytosine (G:C) and adeninepaired with either thymine (A:T), in the case of DNA, or adenine pairedwith uracil (A:U) in the case of RNA.

As used herein, the terms “complementary” or “antisense sequences” meannucleic acid sequences that are substantially complementary over theirentire length and have very few base mismatches. For example, nucleicacid sequences of fifteen bases in length may be termed complementarywhen they have a complementary nucleotide at thirteen or fourteenpositions with only single or double mismatches. Naturally, nucleic acidsequences which are “completely complementary” will be nucleic acidsequences which are entirely complementary throughout their entirelength and have no base mismatches.

While all or part of the gene sequence may be employed in the context ofantisense construction, statistically, any sequence 17 bases long shouldoccur only once in the human genome and, therefore, suffice to specify aunique target sequence. Although shorter oligomers are easier to makeand increase in vivo accessibility, numerous other factors are involvedin determining the specificity of hybridization. Both binding affinityand sequence specificity of an oligonucleotide to its complementarytarget increases with increasing length. It is contemplated thatoligonucleotides of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 ormore base pairs will be used. One can readily determine whether a givenantisense nucleic acid is effective at targeting of the correspondinghost cell gene simply by testing the constructs in vitro to determinewhether the endogenous gene's function is affected or whether theexpression of related genes having complementary sequences is affected.

In certain embodiments, one may wish to employ antisense constructswhich include other elements, for example, those which include C-5propyne pyrimidines. Oligonucleotides which contain C-5 propyneanalogues of uridine and cytidine have been shown to bind RNA with highaffinity and to be potent antisense inhibitors of gene expression(Wagner et al., 1993).

As an alternative to targeted antisense delivery, targeted ribozymes maybe used. The term “ribozyme” refers to an RNA-based enzyme capable oftargeting and cleaving particular base sequences in oncogene DNA andRNA. Ribozymes can either be targeted directly to cells, in the form ofRNA oligo-nucleotides incorporating ribozyme sequences, or introducedinto the cell as an expression construct encoding the desired ribozymalRNA. Ribozymes may be used and applied in much the same way as describedfor antisense nucleic acids.

C) Antigens for Vaccines

Other therapeutics genes might include genes encoding antigens such asviral antigens, bacterial antigens, fungal antigens or parasiticantigens. Viruses include picomavirus, coronavirus, togavirus,flavirvirus, rhabdovirus, paramyxovirus, orthomyxovirus, bunyavirus,arenvirus, reovirus, retrovirus, papovavirus, parvovirus, herpesvirus,poxvirus, hepadnavirus, and spongiform virus. Preferred viral targetsinclude influenza, herpes simplex virus 1 and 2, measles, small pox,polio or HIV. Pathogens include trypanosomes, tapeworms, roundworms,helminths. Also, tumor markers, such as fetal antigen or prostatespecific antigen, may be targeted in this manner. Preferred examplesinclude HIV env proteins and hepatitis B surface antigen. Administrationof a vector according to the present invention for vaccination purposeswould require that the vector-associated antigens be sufficientlynon-immunogenic to enable long term expression of the transgene, forwhich a strong immune response would be desired. Preferably, vaccinationof an individual would only be required infrequently, such as yearly orbiennially, and provide long term immunologic protection against theinfectious agent.

D) Control Regions

In order for the viral vector to effect expression of a transcriptencoding a therapeutic gene, the polynucleotide encoding the therapeuticgene will be under the transcriptional control of a promoter and apolyadenylation signal. A “promoter” refers to a DNA sequence recognizedby the synthetic machinery of the host cell, or introduced syntheticmachinery, that is required to initiate the specific transcription of agene. A polyadenylation signal refers to a DNA sequence recognized bythe synthetic machinery of the host cell, or introduced syntheticmachinery, that is required to direct the addition of a series ofnucleotides on the end of the mRNA transcript for proper processing andtrafficking of the transcript out of the nucleus into the cytoplasm fortranslation. The phrase “under transcriptional control” means that thepromoter is in the correct location in relation to the polynucleotide tocontrol RNA polymerase initiation and expression of the polynucleotide.

The term promoter will be used here to refer to a group oftranscriptional control modules that are clustered around the initiationsite for RNA polymerase II. Much of the thinking about how promoters areorganized derives from analyses of several viral promoters, includingthose for the HSV thymidine kinase (tk) and SV40 early transcriptionunits. These studies, augmented by more recent work, have shown thatpromoters are composed of discrete functional modules, each consistingof approximately 7-20 bp of DNA, and containing one or more recognitionsites for transcriptional activator or repressor proteins.

At least one module in each promoter functions to position the startsite for RNA synthesis. The best known example of this is the TATA box,but in some promoters lacking a TATA box, such as the promoter for themammalian terminal deoxynucleotidyl transferase gene and the promoterfor the SV40 late genes, a discrete element overlying the start siteitself helps to fix the place of initiation.

Additional promoter elements regulate the frequency of transcriptionalinitiation. Typically, these are located in the region 30-110 bpupstream of the start site, although a number of promoters have recentlybeen shown to contain functional elements downstream of the start siteas well. The spacing between promoter elements frequently is flexible,so that promoter function is preserved when elements are inverted ormoved relative to one another. In the tk promoter, the spacing betweenpromoter elements can be increased to 50 bp apart before activity beginsto decline. Depending on the promoter, it appears that individualelements can function either cooperatively or independently to activatetranscription.

The particular promoter that is employed to control the expression of atherapeutic gene is not believed to be critical, so long as it iscapable of expressing the polynucleotide in the targeted cell. Thus,where a human cell is targeted, it is preferable to position thepolynucleotide coding region adjacent to and under the control of apromoter that is capable of being expressed in a human cell. Generallyspeaking, such a promoter might include either a human or viralpromoter. A list of promoters is provided in the Table 2. TABLE 2PROMOTER Immunoglobulin Heavy Chain Immunoglobulin Light Chain T-CellReceptor HLA DQ α and DQ β β-Interferon Interleukin-2 Interleukin-2Receptor MHC Class II 5 MHC Class II HLA-DRα β-Actin Muscle CreatineKinase Prealbumin (Transthyretin) Elastase I Metallothionein CollagenaseAlbumin Gene α-Fetoprotein τ-Globin β-Globin c-fos c-HA-ras InsulinNeural Cell Adhesion Molecule (NCAM) α1-Antitrypsin H2B (TH2B) HistoneMouse or Type I Collagen Glucose-Regulated Proteins (GRP94 and GRP78)Rat Growth Hormone Human Serum Amyloid A (SAA) Troponin I (TN I)Platelet-Derived Growth Factor Duchenne Muscular Dystrophy SV40 PolyomaRetroviruses Papilloma Virus Hepatitis B Virus Human ImmunodeficiencyVirus Cytomegalovirus Gibbon Ape Leukemia Virus

The promoter further may be characterized as an inducible promoter. Aninducible promoter is a promoter which is inactive or exhibits lowactivity except in the presence of an inducer substance. Some examplesof promoters that may be included as a part of the present inventioninclude, but are not limited to, MT II, MMTV, Colleganse, Stromelysin,SV40, Murine MX gene, α-2-Macroglobulin, MHC class I gene h-2 kb, HSP70,Proliferin, Tumor Necrosis Factor, or Thyroid Stimulating Hormone αgene. The associated inducers are shown in Table 3. It is understoodthat any inducible promoter may be used in the practice of the presentinvention and that all such promoters would fall within the spirit andscope of the claimed invention. TABLE 3 Element Inducer MT II PhorbolEster (TPA) Heavy metals MMTV (mouse mammary tumor Glucocorticoidsvirus) β-Interferon poly(rI) × poly (rc) Adenovirus 5 E2 Ela c-junPhorbol Ester (TPA), H₂O₂ Collagenase Phorbol Ester (TPA) StromelysinPhorbol Ester (TPA), IL-1 SV40 Phorbol Ester (TPA) Murine MX GeneInterferon, Newcastle Disease Virus GRP78 Gene A23187 α-2-MacroglobulinIL-6 Vimentin Serum MHC Class I Gene H-2 kB Interferon HSP70 Ela, SV40Large T Antigen Proliferin Phorbol Ester-TPA Tumor Necrosis Factor FMAThyroid Stimulating Hormone α Thyroid Hormone Gene

In various embodiments, the human cytomegalovirus (CMV) immediate earlygene promoter, the SV40 early promoter and the Rous sarcoma virus longterminal repeat can be used to obtain high-level expression of thepolynucleotide of interest. The use of other viral or mammalian cellularor bacterial phage promoters which are well-known in the art to achieveexpression of polynucleotides is contemplated as well, provided that thelevels of expression are sufficient to produce a growth inhibitoryeffect.

By employing a promoter with well-known properties, the level andpattern of expression of a polynucleotide following transfection can beoptimized. For example, selection of a promoter which is active inspecific cells, such as tyrosinase (melanoma), alpha-fetoprotein andalbumin (liver tumors), CC10 (lung tumor) and prostate-specific antigen(prostate tumor) will permit tissue-specific expression of thetherapeutic gene.

Enhancers were originally detected as genetic elements that increasedtranscription from a promoter located at a distant position on the samemolecule of DNA. This ability to act over a large distance had littleprecedent in classic studies of prokaryotic transcriptional regulation.Subsequent work showed that regions of DNA with enhancer activity areorganized much like promoters. That is, they are composed of manyindividual elements, each of which binds to one or more transcriptionalproteins.

The basic distinction between enhancers and promoters is operational. Anenhancer region as a whole must be able to stimulate transcription at adistance; this need not be true of a promoter region or its componentelements. On the other hand, a promoter must have one or more elementsthat direct initiation of RNA synthesis at a particular site and in aparticular orientation, whereas enhancers lack these specificities.Promoters and enhancers are often overlapping and contiguous, oftenseeming to have a very similar modular organization.

Additionally any promoter/enhancer combination (as per the EukaryoticPromoter Data Base (EPDB)) could also be used to drive expression of aparticular construct. Use of a T3, T7 or SP6 cytoplasmic expressionsystem is another possible embodiment. Eukaryotic cells can supportcytoplasmic transcription from certain bacteriophage promoters if theappropriate bacteriophage polymerase is provided, either as part of thedelivery complex or as an additional genetic expression vector.

Where a cDNA insert is employed, one will typically desire to include apolyadenylation signal to effect proper polyadenylation of the genetranscript. The nature of the polyadenylation signal is not believed tobe crucial to the successful practice of the invention, and any suchsequence may be employed. Such polyadenylation signals as that fromSV40, bovine growth hormone, and the herpes simplex virus thymidinekinase gene have been found to function well in a number of targetcells.

7. METHODS OF GENE TRANSFER

In order to create the helper cell lines of the present invention, andto create recombinant adenovirus vectors for use therewith, variousgenetic (i.e. DNA) constructs must be delivered to a cell. One way toachieve this is via viral transductions using infectious viralparticles, for example, by transformation with an adenovirus vector ofthe present invention. Alternatively, retroviral or bovine papillomavirus may be employed, both of which permit permanent transformation ofa host cell with a gene(s) of interest. In other situations, the nucleicacid to be transferred is not infectious, i.e., contained in aninfectious virus particle. This genetic material must rely on non-viralmethods for transfer.

Several non-viral methods for the transfer of expression constructs intocultured mammalian cells also are contemplated by the present invention.These include calcium phosphate precipitation (Graham and Van Der Eb,1973; Chen and Okayama, 1987; Rippe et al., 1990) DEAE-dextran (Gopal,1985), electroporation (Tur-Kaspa et al., 1986; Potter et al., 1984),direct microinjection (Harland and Weintraub, 1985), DNA-loadedliposomes (Nicolau and Sene, 1982; Fraley et al., 1979), cell sonication(Fechheimer et al., 1987), gene bombardment using high velocitymicroprojectiles (Yang et al., 1990), and receptor-mediated transfection(Wu and Wu, 1987; Wu and Wu, 1988).

Once the construct has been delivered into the cell the nucleic acidencoding the therapeutic gene may be positioned and expressed atdifferent sites. In certain embodiments, the nucleic acid encoding thetherapeutic gene may be stably integrated into the genome of the cell.This integration may be in the cognate location and orientation viahomologous recombination (gene replacement) or it may be integrated in arandom, non-specific location (gene augmentation). In yet furtherembodiments, the nucleic acid may be stably maintained in the cell as aseparate, episomal segment of DNA. Such nucleic acid segments or“episomes” encode sequences sufficient to permit maintenance andreplication independent of or in synchronization with the host cellcycle. How the expression construct is delivered to a cell and where inthe cell the nucleic acid remains is dependent on the type of expressionconstruct employed.

In one embodiment of the invention, the expression construct may simplyconsist of naked recombinant DNA or plasmids. Transfer of the constructmay be performed by any of the methods mentioned above which physicallyor chemically permeabilize the cell membrane. This is particularityapplicable for transfer in vitro, however, it may be applied for in vivouse as well. Dubensky et al. (1984) successfully injected polyomavirusDNA in the form of CaPO₄ precipitates into liver and spleen of adult andnewborn mice demonstrating active viral replication and acute infection.Benvenisty and Neshif (1986) also demonstrated that directintraperitoneal injection of CaPO₄ precipitated plasmids results inexpression of the transfected genes. It is envisioned that DNA encodinga CAM may also be transferred in a similar manner in vivo and expressCAM.

Another embodiment of the invention for transferring a naked DNAexpression construct into cells may involve particle bombardment. Thismethod depends on the ability to accelerate DNA coated microprojectilesto a high velocity allowing them to pierce cell membranes and entercells without killing them (Klein et al., 1987). Several devices foraccelerating small particles have been developed. One such device relieson a high voltage discharge to generate an electrical current, which inturn provides the motive force (Yang et al., 1990). The microprojectilesused have consisted of biologically inert substances such as tungsten orgold beads.

In a further embodiment of the invention, the expression construct maybe entrapped in a liposome. Liposomes are vesicular structurescharacterized by a phospholipid bilayer membrane and an inner aqueousmedium. Multilamellar liposomes have multiple lipid layers separated byaqueous medium. They form spontaneously when phospholipids are suspendedin an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers (Ghosh andBachhawat, 1991).

Liposome-mediated nucleic acid delivery and expression of foreign DNA invitro has been very successful. Using the β-lactamase gene, Wong et al.(1980) demonstrated the feasibility of liposome-mediated delivery andexpression of foreign DNA in cultured chick embryo, HeLa, and hepatomacells. Nicolau et al. (1987) accomplished successful liposome-mediatedgene transfer in rats after intravenous injection. Also included arevarious commercial approaches involving “lipofection” technology.

In certain embodiments of the invention, the liposome may be complexedwith a hemagglutinating virus (HVJ). This has been shown to facilitatefusion with the cell membrane and promote cell entry ofliposome-encapsulated DNA (Kaneda et al., 1989). In other embodiments,the liposome may be complexed or employed in conjunction with nuclearnonhistone chromosomal proteins (HMG-1) (Kato et al., 1991). In yetfurther embodiments, the liposome may be complexed or employed inconjunction with both HVJ and HMG-1. In that such expression constructshave been successfully employed in transfer and expression of nucleicacid in vitro and in vivo, then they are applicable for the presentinvention.

Other expression constructs which can be employed to deliver a nucleicacid encoding a therapeutic gene into cells are receptor-mediateddelivery vehicles. These take advantage of the selective uptake ofmacromolecules by receptor-mediated endocytosis in almost all eukaryoticcells. Because of the cell type-specific distribution of variousreceptors, the delivery can be highly specific (Wu and Wu, 1993).

Receptor-mediated gene targeting vehicles generally consist of twocomponents: a cell receptor-specific ligand and a DNA-binding agent.Several ligands have been used for receptor-mediated gene transfer. Themost extensively characterized ligands are asialoorosomucoid (ASOR) (Wuand Wu, 1987) and transferring (Wagner et al., 1990). Recently, asynthetic neoglycoprotein, which recognizes the same receptor as ASOR,has been used as a gene delivery vehicle (Ferkol et al., 1993; Peraleset al., 1994) and epidermal growth factor (EGF) has also been used todeliver genes to squamous carcinoma cells (Myers, EPO 0273085).

In other embodiments, the delivery vehicle may comprise a ligand and aliposome. For example, Nicolau et al. (1987) employed lactosyl-ceramide,a galactose-terminal asialganglioside, incorporated into liposomes andobserved an increase in the uptake of the insulin gene by hepatocytes.Thus, it is feasible that a nucleic acid encoding a therapeutic genealso may be specifically delivered into a cell type such as prostate,epithelial or tumor cells, by any number of receptor-ligand systems withor without liposomes. For example, the human prostate-specific antigen(Watt et al., 1986) may be used as the receptor for mediated delivery ofa nucleic acid in prostate tissue.

8. REMOVING NUCLEIC ACID CONTAMINANTS

The present invention employs nucleases to remove contaminating nucleicacids. Exemplary nucleases include Benzonase®, Pulmozyme®; or any otherDNase or RNase commonly used within the art.

Enzymes such as Benzonaze® degrade nucleic acid and have no proteolyticactivity. The ability of Benzonase® to rapidly hydrolyze nucleic acidsmakes the enzyme ideal for reducing cell lysate viscosity. It is wellknown that nucleic acids may adhere to cell derived particles such asviruses. The adhesion may interfere with separation due toagglomeration, change in size of the particle or change in particlecharge, resulting in little if any product being recovered with a givenpurification scheme. Benzonase® is well suited for reducing the nucleicacid load during purification, thus eliminating the interference andimproving yield.

As with all endonucleases, Benzonase® hydrolyzes internal phosphodiesterbonds between specific nucleotides. Upon complete digestion, all freenucleic acids present in solution are reduced to oligonucleotides 2 to 4bases in length.

9. PURIFICATION TECHNIQUES

The present invention employs a number of different purification topurify adenoviral vectors of the present invention. Such techniquesinclude those based on sedimentation and chromatography and aredescribed in more detail herein below.

A) Density Gradient Centrifugation

There are two methods of density gradient centrifugation, the rate zonaltechnique and the isopycnic (equal density) technique, and both can beused when the quantitative separation of all the components of a mixtureof particles is required. They are also used for the determination ofbuoyant densities and for the estimation of sedimentation coefficients.

Particle separation by the rate zonal technique is based upondifferences in size or sedimentation rates. The technique involvescarefully layering a sample solution on top of a performed liquiddensity gradient, the highest density of which exceeds that of thedensest particles to be separated. The sample is then centrifuged untilthe desired degree of separation is effected, i.e., for sufficient timefor the particles to travel through the gradient to form discrete zonesor bands which are spaced according to the relative velocities of theparticles. Since the technique is time dependent, centrifugation must beterminated before any of the separated zones pellet at the bottom of thetube. The method has been used for the separation of enzymes, hormones,RNA-DNA hybrids, ribosomal subunits, subcellular organelles, for theanalysis of size distribution of samples of polysomes and forlipoprotein fractionations.

The sample is layered on top of a continuous density gradient whichspans the whole range of the particle densities which are to beseparated. The maximum density of the gradient, therefore, must alwaysexceed the density of the most dense particle. During centrifugation,sedimentation of the particles occurs until the buoyant density of theparticle and the density of the gradient are equal (i.e., wherep_(p)=p_(m) in equation 2.12). At this point no further sedimentationoccurs, irrespective of how long centrifugation continues, because theparticles are floating on a cushion of material that has a densitygreater than their own.

Isopycnic centrifugation, in contrast to the rate zonal technique, is anequilibrium method, the particles banding to form zones each at theirown characteristic buoyant density. In cases where, perhaps, not all thecomponents in a mixture of particles are required, a gradient range canbe selected in which unwanted components of the mixture will sediment tothe bottom of the centrifuge tube whilst the particles of interestsediment to their respective isopycnic positions. Such a techniqueinvolves a combination of both the rate zonal and isopycnic approaches.

Isopycnic centrifugation depends solely upon the buoyant density of theparticle and not its shape or size and is independent of time. Hencesoluble proteins, which have a very similar density (e.g., p=1.3 g cm⁻³in sucrose solution), cannot usually be separated by this method,whereas subcellular organelles (e.g., Golgi apparatus, p=1.11 g cm⁻³,mitochondria, p=1.19 g cm⁻³ and peroxisomes, p=1.23 g cm⁻³ in sucrosesolution) can be effectively separated.

As an alternative to layering the particle mixture to be separated ontoa preformed gradient, the sample is initially mixed with the gradientmedium to give a solution of uniform density, the gradient‘self-forming’, by sedimentation equilibrium, during centrifugation. Inthis method (referred to as the equilibrium isodensity method), use isgenerally made of the salts of heavy metals (e.g., caesium or rubidium),sucrose, colloidal silica or Metrizamide.

The sample (e.g., DNA) is mixed homogeneously with, for example, aconcentrated solution of caesium chloride. Centrifugation of theconcentrated caesium chloride solution results in the sedimentation ofthe CsCl molecules to form a concentration gradient and hence a densitygradient. The sample molecules (DNA), which were initially uniformlydistributed throughout the tube now either rise or sediment until theyreach a region where the solution density is equal to their own buoyantdensity, i.e. their isopycnic position, where they will band to formzones. This technique suffers from the disadvantage that often very longcentrifugation times (e.g., 36 to 48 hours) are required to establishequilibrium. However, it is commonly used in analytical centrifugationto determine the buoyant density of a particle, the base composition ofdouble stranded DNA and to separate linear from circular forms of DNA.

Many of the separations can be improved by increasing the densitydifferences between the different forms of DNA by the incorporation ofheavy isotopes (e.g., ¹⁵N) during biosynthesis, a technique used byLeselson and Stahl to elucidate the mechanism of DNA replication inEscherichia coli, or by the binding of heavy metal ions or dyes such asethidium bromide. Isopycnic gradients have also been used to separateand purify viruses and analyze human plasma lipoproteins.

B) Chromatography

In certain embodiments of the invention, it will be desirable to producepurified adenovirus. Purification techniques are well known to those ofskill in the art. These techniques tend to involve the fractionation ofthe cellular milieu to separate the adenovirus particles from othercomponents of the mixture. Having separated adenoviral particles fromthe other components, the adenovirus may be purified usingchromatographic and electrophoretic techniques to achieve completepurification. Analytical methods particularly suited to the preparationof a pure adenoviral particle of the present invention are ion-exchangechromatography, size exclusion chromatography; polyacrylamide gelelectrophoresis. A particularly efficient purification method to beemployed in conjunction with the present invention is HPLC.

Certain aspects of the present invention concern the purification, andin particular embodiments, the substantial purification, of anadenoviral particle. The term “purified” as used herein, is intended torefer to a composition, isolatable from other components, wherein theadenoviral particle is purified to any degree relative to itsnaturally-obtainable form. A purified adenoviral particle therefore alsorefers to an adenoviral component, free from the environment in which itmay naturally occur.

Generally, “purified” will refer to an adenoviral particle that has beensubjected to fractionation to remove various other components, and whichcomposition substantially retains its expressed biological activity.Where the term “substantially purified” is used, this designation willrefer to a composition in which the particle, protein or peptide formsthe major component of the composition, such as constituting about 50%or more of the constituents in the composition.

Various methods for quantifying the degree of purification of a proteinor peptide will be known to those of skill in the art in light of thepresent disclosure. These include, for example, determining the specificactivity of an active fraction, or assessing the amount of polypeptideswithin a fraction by SDS/PAGE analysis. A preferred method for assessingthe purity of a fraction is to calculate the specific activity of thefraction, to compare it to the specific activity of the initial extract,and to thus calculate the degree of purity, herein assessed by a “-foldpurification number”. The actual units used to represent the amount ofactivity will, of course, be dependent upon the particular assaytechnique chosen to follow the purification and whether or not theexpressed protein or peptide exhibits a detectable activity.

There is no general requirement that the adenovirus, always be providedin their most purified state. Indeed, it is contemplated that lesssubstantially purified products will have utility in certainembodiments. Partial purification may be accomplished by using fewerpurification steps in combination, or by utilizing different forms ofthe same general purification scheme. For example, it is appreciatedthat a cation-exchange column chromatography performed utilizing an HPLCapparatus will generally result in a greater-fold purification than thesame technique utilizing a low pressure chromatography system. Methodsexhibiting a lower degree of relative purification may have advantagesin total recovery of protein product, or in maintaining the activity ofan expressed protein.

Of course, it is understood that the chromatographic techniques andother purification techniques known to those of skill in the art mayalso be employed to purify proteins expressed by the adenoviral vectorsof the present invention. Ion exchange chromatography and highperformance liquid chromatography are exemplary purification techniquesemployed in the purification of adenoviral particles and are describedin further detail herein below.

Ion-Exchange Chromatography. The basic principle of ion-exchangechromatography is that the affinity of a substance for the exchangerdepends on both the electrical properties of the material and therelative affinity of other charged substances in the solvent. Hence,bound material can be eluted by changing the pH, thus altering thecharge of the material, or by adding competing materials, of which saltsare but one example. Because different substances have differentelectrical properties, the conditions for release vary with each boundmolecular species. In general, to get good separation, the methods ofchoice are either continuous ionic strength gradient elution or stepwiseelution. (A gradient of pH alone is not often used because it isdifficult to set up a pH gradient without simultaneously increasingionic strength.) For an anion exchanger, either pH and ionic strengthare gradually increased or ionic strength alone is increased. For acation exchanger, both pH and ionic strength are increased. The actualchoice of the elution procedure is usually a result of trial and errorand of considerations of stability. For example, for unstable materials,it is best to maintain fairly constant pH.

An ion exchanger is a solid that has chemically bound charged groups towhich ions are electrostatically bound; it can exchange these ions forions in aqueous solution. Ion exchangers can be used in columnchromatography to separate molecules according to charge; actually otherfeatures of the molecule are usually important so that thechromatographic behavior is sensitive to the charge density, chargedistribution, and the size of the molecule.

The principle of ion-exchange chromatography is that charged moleculesadsorb to ion exchangers reversibly so that molecules can be bound oreluted by changing the ionic environment. Separation on ion exchangersis usually accomplished in two stages: first, the substances to beseparated are bound to the exchanger, using conditions that give stableand tight binding; then the column is eluted with buffers of differentpH, ionic strength, or composition and the components of the buffercompete with the bound material for the binding sites.

An ion exchanger is usually a three-dimensional network or matrix thatcontains covalently linked charged groups. If a group is negativelycharged, it will exchange positive ions and is a cation exchanger. Atypical group used in cation exchangers is the sulfonic group, SO₃ ⁻. Ifan H⁺ is bound to the group, the exchanger is said to be in the acidform; it can, for example, exchange on H⁺ for one Na⁺ or two H⁺ for oneCa₂ ⁺. The sulfonic acid group is called a strongly acidic cationexchanger. Other commonly used groups are phenolic hydroxyl andcarboxyl, both weakly acidic cation exchangers. If the charged group ispositive—for example, a quaternary amino group—it is a strongly basicanion exchanger. The most common weakly basic anion exchangers arearomatic or aliphatic amino groups.

The matrix can be made of various material. Commonly used materials aredextran, cellulose, agarose and copolymers of styrene and vinylbenzenein which the divinylbenzene both cross-links the polystyrene strands andcontains the charged groups. Table 4 gives the composition of many ionexchangers.

The total capacity of an ion exchanger measures its ability to take upexchangeable groups per milligram of dry weight. This number is suppliedby the manufacturer and is important because, if the capacity isexceeded, ions will pass through the column without binding. TABLE 4Matrix Exchanger Functional Group Tradename Dextran Strong CationicSulfopropyl SP-Sephadex Weak Cationic Carboxymethyl CM-Sephadex StrongAnionic Diethyl-(2- QAE-Sephadex hydroxypropyl)- aminoethyl Weak AnionicDiethylaminoethyl DEAE-Sephadex Cellulose Cationic CarboxymethylCM-Cellulose Cationic Phospho P-cel Anionic DiethylaminoethylDEAE-cellulose Anionic Polyethylenimine PEI-Cellulose AnionicBenzoylated- DEAE(BND)-cellulose naphthoylated, deiethylaminoethylAnionic p-Aminobenzyl PAB-cellulose Styrene- Strong Cationic Sulfonicacid AG 50 divinyl- Strong Anionic AG 1 benzene Strong Cationic + StrongSulfonic acid + AG 501 Anionic Tetramethylammonium Acrylic Weak CationicCarboxylic Bio-Rex 70 Phenolic Strong Cationic Sulfonic acid Bio-Rex 40Expoxyamine Weak Anionic Tertiary amino AG-3

The available capacity is the capacity under particular experimentalconditions (i.e., pH, ionic strength). For example, the extent to whichan ion exchanger is charged depends on the pH (the effect of pH issmaller with strong ion exchangers). Another factor is ionic strengthbecause small ions near the charged groups compete with the samplemolecule for these groups. This competition is quite effective if thesample is a macromolecule because the higher diffusion coefficient ofthe small ion means a greater number of encounters. Clearly, as bufferconcentration increases, competition becomes keener.

The porosity of the matrix is an important feature because the chargedgroups are both inside and outside the matrix and because the matrixalso acts as a molecular sieve. Large molecules may be unable topenetrate the pores; so the capacity will decease with increasingmolecular dimensions. The porosity of the polystyrene-based resins isdetermined by the amount of cross-linking by the divinylbenzene(porosity decreases with increasing amounts of divinylbenzene). With theDowex and AG series, the percentage of divinylbenzene is indicated by anumber after an X—hence, Dowex 50-X8 is 8% divinylbenzene

Ion exchangers come in a variety of particle sizes, called mesh size.Finer mesh means an increased surface-to-volume ration and thereforeincreased capacity and decreased time for exchange to occur for a givenvolume of the exchanger. On the other hand, fine mesh means a slow flowrate, which can increase diffusional spreading. The use of very fineparticles, approximately 10 μm in diameter and high pressure to maintainan adequate flow is called high-performance or high-pressure liquidchromatography or simply HPLC.

Such a collection of exchangers having such different properties—charge,capacity, porosity, mesh—makes the selection of the appropriate one foraccomplishing a particular separation difficult. How to decide on thetype of column material and the conditions for binding and elution isdescribed in the following Examples.

There are a number of choice to be made when employing ion exchangechromatography as a technique. The first choice to be made is whetherthe exchanger is to be anionic or cationic. If the materials to be boundto the column have a single charge (i.e., either plus or minus), thechoice is clear. However, many substances (e.g., proteins, viruses),carry both negative and positive charges and the net charge depends onthe pH. In such cases, the primary factor is the stability of thesubstance at various pH values. Most proteins have a pH range ofstability (i.e., in which they do not denature) in which they are eitherpositively or negatively charged. Hence, if a protein is stable at pHvalues above the isoelectric point, an anion exchanger should be used;if stable at values below the isoelectric point, a cation exchanger isrequired.

The choice between strong and weak exchangers is also based on theeffect of pH on charge and stability. For example, if a weakly ionizedsubstance that requires very low or high pH for ionization ischromatographed, a strong ion exchanger is called for because itfunctions over the entire pH range. However, if the substance is labile,weak ion exchangers are preferable because strong exchangers are oftencapable of distorting a molecule so much that the molecule denatures.The pH at which the substance is stable must, of course, be matched tothe narrow range of pH in which a particular weak exchanger is charged.Weak ion exchangers are also excellent for the separation of moleculeswith a high charge from those with a small charge, because the weaklycharged ions usually fail to bind. Weak exchangers also show greaterresolution of substances if charge differences are very small. If amacromolecule has a very strong charge, it may be impossible to elutefrom a strong exchanger and a weak exchanger again may be preferable. Ingeneral, weak exchangers are more useful than strong exchangers.

The Sephadex and Bio-gel exchangers offer a particular advantage formacromolecules that are unstable in low ionic strength. Because thecross-links in these materials maintain the insolubility of the matrixeven if the matrix is highly polar, the density of ionizable groups canbe made several times greater than is possible with cellulose ionexchangers. The increased charge density means increased affinity sothat adsorption can be carried out at higher ionic strengths. On theother hand, these exchangers retain some of their molecular sievingproperties so that sometimes molecular weight differences annul thedistribution caused by the charge differences; the molecular sievingeffect may also enhance the separation.

Small molecules are best separated on matrices with small pore size(high degree of cross-linking) because the available capacity is large,whereas macromolecules need large pore size. However, except for theSephadex type, most ion exchangers do not afford the opportunity formatching the porosity with the molecular weight.

The cellulose ion exchangers have proved to be the best for purifyinglarge molecules such as proteins and polynucleotides. This is becausethe matrix is fibrous, and hence all functional groups are on thesurface and available to even the largest molecules. In may caseshowever, beaded forms such as DEAE-Sephacel and DEAE-Biogel P are moreuseful because there is a better flow rate and the molecular sievingeffect aids in separation.

Selecting a mesh size is always difficult. Small mesh size improvesresolution but decreases flow rate, which increases zone spreading anddecreases resolution. Hence, the appropriate mesh size is usuallydetermined empirically.

Because buffers themselves consist of ions, they can also exchange, andthe pH equilibrium can be affected. To avoid these problems, the rule ofbuffers is adopted: use cationic buffers with anion exchangers andanionic buffers with cation exchangers. Because ionic strength is afactor in binding, a buffer should be chosen that has a high bufferingcapacity so that its ionic strength need not be too high. Furthermore,for best resolution, it has been generally found that the ionicconditions used to apply the sample to the column (the so-calledstarting conditions) should be near those used for eluting the column.

High Performance Liquid Chromatography (HPLC) is characterized by a veryrapid separation with extraordinary resolution of peaks. This isachieved by the use of very fine particles and high pressure to maintainan adequate flow rate. Separation can be accomplished in a matter ofminutes, or at most an hour. Moreover, only a very small volume of thesample is needed because the particles are so small and close-packedthat the void volume is a very small fraction of the bed volume. Also,the concentration of the sample need not be very great because the bandsare so narrow that there is very little dilution of the sample.

10. Pharmaceutical Compositions and Formulations

When purified according to the methods set forth above, the viralparticles of the present invention will be administered, in vitro, exvivo or in vivo is contemplated. Thus, it will be desirable to preparethe complex as a pharmaceutical composition appropriate for the intendedapplication. Generally this will entail preparing a pharmaceuticalcomposition that is essentially free of pyrogens, as well as any otherimpurities that could be harmful to humans or animals. One also willgenerally desire to employ appropriate salts and buffers to render thecomplex stable and allow for complex uptake by target cells.

Aqueous compositions of the present invention comprise an effectiveamount of the expression construct and nucleic acid, dissolved ordispersed in a pharmaceutically acceptable carrier or aqueous medium.Such compositions can also be referred to as inocula. The phrases“pharmaceutically or pharmacologically acceptable” refer to molecularentities and compositions that do not produce an adverse, allergic orother untoward reaction when administered to an animal, or a human, asappropriate. As used herein, “pharmaceutically acceptable carrier”includes any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents and thelike. The use of such media and agents for pharmaceutical activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active ingredient, its use inthe therapeutic compositions is contemplated. Supplementary activeingredients also can be incorporated into the compositions.

Solutions of the active compounds as free base or pharmacologicallyacceptable salts can be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions also can beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The viral particles of the present invention may include classicpharmaceutical preparations for use in therapeutic regimens, includingtheir administration to humans. Administration of therapeuticcompositions according to the present invention will be via any commonroute so long as the target tissue is available via that route. Thisincludes oral, nasal, buccal, rectal, vaginal or topical. Alternatively,administration will be by orthotopic, intradermal subcutaneous,intramuscular, intraperitoneal, or intravenous injection. Suchcompositions would normally be administered as pharmaceuticallyacceptable compositions that include physiologically acceptablecarriers, buffers or other excipients. For application against tumors,direct intratumoral injection, inject of a resected tumor bed, regional(i.e., lymphatic) or general administration is contemplated. It also maybe desired to perform continuous perfusion over hours or days via acatheter to a disease site, e.g., a tumor or tumor site.

The therapeutic compositions of the present invention are advantageouslyadministered in the form of injectable compositions either as liquidsolutions or suspensions; solid forms suitable for solution in, orsuspension in, liquid prior to injection may also be prepared. Thesepreparations also may be emulsified. A typical composition for suchpurpose comprises a pharmaceutically acceptable carrier. For instance,the composition may contain about 100 mg of human serum albumin permilliliter of phosphate buffered saline. Other pharmaceuticallyacceptable carriers include aqueous solutions, non-toxic excipients,including salts, preservatives, buffers and the like may be used.Examples of non-aqueous solvents are propylene glycol, polyethyleneglycol, vegetable oil and injectable organic esters such as ethyloleate.Aqueous carriers include water, alcoholic/aqueous solutions, salinesolutions, parenteral vehicles such as sodium chloride, Ringer'sdextrose, etc. Intravenous vehicles include fluid and nutrientreplenishers. Preservatives include antimicrobial agents, anti-oxidants,chelating agents and inert gases. The pH and exact concentration of thevarious components the pharmaceutical composition are adjusted accordingto well known parameters.

Additional formulations which are suitable for oral administration. Oralformulations include such typical excipients as, for example,pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate and the like. Thecompositions take the form of solutions, suspensions, tablets, pills,capsules, sustained release formulations or powders. When the route istopical, the form may be a cream, ointment, salve or spray.

An effective amount of the therapeutic agent is determined based on theintended goal, for example (i) inhibition of tumor cell proliferation,(ii) elimination or killing of tumor cells, (iii) vaccination, or (iv)gene transfer for long term expression of a therapeutic gene. The term“unit dose” refers to physically discrete units suitable for use in asubject, each unit containing a predetermined-quantity of thetherapeutic composition calculated to produce the desired responses,discussed above, in association with its administration, i.e., theappropriate route and treatment regimen. The quantity to beadministered, both according to number of treatments and unit dose,depends on the subject to be treated, the state of the subject and theresult desired. Multiple gene therapeutic regimens are expected,especially for adenovirus.

In certain embodiments of the present invention, an adenoviral vectorencoding a tumor suppressor gene will be used to treat cancer patients.Typical amounts of an adenovirus vector used in gene therapy of canceris 10³-10¹⁵ PFU/dose, (10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹,10¹², 10¹³, 10¹⁴, 10¹⁵) wherein the dose may be divided into severalinjections at different sites within a solid tumor. The treatmentregimen also may involve several cycles of administration of the genetransfer vector over a period of 3-10 weeks. Administration of thevector for longer periods of time from months to years may be necessaryfor continual therapeutic benefit.

In another embodiment of the present invention, an adenoviral vectorencoding a therapeutic gene may be used to vaccinate humans or othermammals. Typically, an amount of virus effective to produce the desiredeffect, in this case vaccination, would be administered to a human ormammal so that long term expression of the transgene is achieved and astrong host immune response develops. It is contemplated that a seriesof injections, for example, a primary injection followed by two boosterinjections, would be sufficient to induce an long term immune response.A typical dose would be from 10⁶ to 10¹⁵ PFU/injection depending on thedesired result. Low doses of antigen generally induce a strongcell-mediated response, whereas high doses of antigen generally inducean antibody-mediated immune response. Precise amounts of the therapeuticcomposition also depend on the judgment of the practitioner and arepeculiar to each individual.

11. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Materials and Methods

A) Cells

293 cells (human epithelial embryonic kidney cells) from the Master CellBank were used for the studies.

B) Media

Dulbecco's modified Eagle's medium (DMEM, 4.5 g/L glucose)+10% fetalbovine serum (FBS) was used for the cell growth phase. For the virusproduction phase, the FBS concentration in DMEM was lowered to 2%.

C) Virus

AdCMVp53 is a genetically engineered, replication-incompetent human type5 adenovirus expressing the human wild type p53 protein under control ofthe cytomegalovirus (CMV) immediate early promoter.

D) Celligen Bioreactor

A Celligen bioreactor (New Brunswick Scientific, Co. Inc.) with 5 Ltotal volume (3.5 L working volume) was used to produce virussupernatant using microcarrier culture. 13 g/L glass coated microcarrier(SoloHill) was used for culturing cells in the bioreactor.

E) Production of Virus Supernatant in the Celligen Bioreactor

293 cells from master cell bank (MCB) were thawed and expanded intoCellfactories (Nunc). Cells were generally split at a confluence ofabout 85-90%. Cells were inoculated into the bioreactor at aninoculation concentration of 1×10⁵ cells/ml. Cells were allowed toattach to the microcarriers by intermittent agitation. Continuousagitation at a speed of 30 rpm was started 6-8 hr post cell inoculation.Cells were cultured for 7 days with process parameters set at pH=7.20,dissolved oxygen (DO)=60% of air saturation, temperature=37° C. On day8, cells were infected with AdCMVp53 at an MOI of 5. Fifty hr post virusinfection, agitation speed was increased from 30 rpm to 150 rpm tofacilitate cell lysis and release of the virus into the supernatant. Thevirus supernatant was harvested 74 hr post-infection. The virussupernatant was then filtered for further concentration/diafiltration.

F) Cellcube™ Bioreactor System

A Cellcube™ bioreactor system (Corning-Costar) was also used for theproduction of AdCMVp53 virus. It is composed of a disposable cellculture module, an oxygenator, a medium recirculation pump and a mediumpump for perfusion. The cell culture module used has a culture surfacearea of 21,550 cm² (1 mer).

G) Production of Virus in the Cellcube™

293 cells from master cell bank (MCB) were thawed and expanded intoCellfactories (Nunc). Cells were generally split at a confluence ofabout 85-90%. Cells were inoculated into the Cellcube™ according to themanufacturer's recommendation. Inoculation cell densities were in therange of 1-1.5×10⁴/cm². Cells were allowed to grow for 7 days at 37° C.under culture conditions of pH=7.20, DO=60% air saturation. Mediumperfusion rate was regulated according to the glucose concentration inthe Cellcube™. One day before viral infection, medium for perfusion waschanged from DMEM+10% FBS to DMEM+2% FBS. On day 8, cells were infectedwith AdCMVp53 virus at a multiplicity of infection (MOI) of 5. Mediumperfusion was stopped for 1 hr immediately after infection then resumedfor the remaining period of the virus production phase. Culture washarvested 45-48 hr post-infection.

H) Lysis Solution

Tween-20 (Fisher Chemicals) at a concentration of 1% (v/v) in 20 mMTris+0.25 M NaCl+1 mM MgCl₂, pH=7.50 buffer was used to lyse cells atthe end of the virus production phase in the Cellcube™.

I) Clarification and Filtration

Virus supernatant from the Celligen bioreactor and virus solution fromthe Cellcube™ were first clarified using a depth filter (Preflow,GelmanSciences), then was filtered through a 0.8/0.22 μm filter (SuporCap 100, GelmanSciences).

J) Concentration/Diafiltration

Tangential flow filtration (TFF) was used to concentrate and bufferexchange the virus supernatant from the Celligen bioreactor and thevirus solution from the Cellcube™. A Pellicon II mini cassette(Millipore) of 300 K nominal molecular weight cut off (NMWC) was usedfor the concentration and diafiltration. Virus solution was firstconcentrated 10-fold. This was followed by 4 sample volume of bufferexchange against 20 mM Tris+1.0 M NaCl+1 mM MgCl₂, pH=9.00 buffer usingthe constant volume diafiltration method.

Similar concentration/diafiltration was carried out for the columnpurified virus. A Pellicon II mini cassette of 100 K NMWC was usedinstead of the 300 K NMWC cassette. Diafiltration was done against 20 mMTris+0.25 M NaCl+1 mM MgCl₂, pH=9.00 buffer or Dulbecco's phosphatebuffered saline (DPBS).

K) Benzonase Treatment

The concentrated/diafiltrated virus solution was treated with Benzonase™(American International Chemicals) at a concentration of 100 u/ml, roomtemperature overnight to reduce the contaminating nucleic acidconcentration in the virus solution.

L) CsCl Gradient Ultracentrifugation

Crude virus solution was purified using double CsCl gradientultracentrifugation using a SW40 rotor in a Beckman ultracentrifuge(XL-90). First, 7 ml of crude virus solution was overlaid on top of astep CsCl gradient made of equal volume of 2.5 ml of 1.25 g/ml and 1.40g/ml CsCl solution, respectively. The CsCl gradient was centrifuged at35,000 rpm for 1 hr at room temperature. The virus band at the gradientinterface was recovered. The recovered virus was then further purifiedthrough a isopicnic CsCl gradient. This was done by mixing the virussolution with at least 1.5-fold volume of 1.33 g/ml CsCl solution. TheCsCl solution was centrifuged at 35,000 rpm for at least 18 hr at roomtemperature. The lower band was recovered as the intact virus. The viruswas immediately dialyzed against 20 mM Tris+1 mM MgCl₂, pH=7.50 bufferto remove CsCl. The dialyzed virus was stored at −70° C. for future use.

M) Ion Exchange Chromatography (IEC) Purification

The Benzonase treated virus solution was purified using IEC. Stronganionic resin Toyopearl SuperQ 650M (Tosohaas) was used for thepurification. A FPLC system (Pharmacia) with a XK16 column (Pharmacia)were used for the initial method development. Further scale-up studieswere carried out using a BioPilot system (Pharmacia) with a XK 50 column(Pharmacia). Briefly, the resin was packed into the columns andsanitized with 1 N NaOH, then charged with buffer B which was followedby conditioning with buffer A. Buffers A and B were composed of 20 mMTris+0.25 M NaCl+1 mM MgCl₂, pH=9.00 and 20 mM Tris+2M NaCl+1 mM MgCl₂,pH=9.00, respectively. Viral solution sample was loaded onto theconditioned column, followed by washing the column with buffer A untilthe UV absorption reached base line. The purified virus was eluted fromthe column by using a 10 column volume of linear NaCl gradient.

N) HPLC Analysis

A HPLC analysis procedure was developed for evaluating the efficiency ofvirus production and purification. Tris(hydroxymethyl)aminomethane(tris) was obtained from FisherBiotech (Cat# BP154-1; Fair Lawn, N.J.,U.S.A.); sodium chloride (NaCl) was obtained from Sigma (Cat# S-7653,St. Louis, Mo., U.S.A.). Both were used directly without furtherpurification. HPLC analyses were performed on an Analytical GradientSystem from Beckman, with Gold Workstation Software (126 binary pump and168 diode array detector) equipped with an anion-exchange column fromTosoHaas (7.5 cm×7.5 mm ID, 10 μm particle size, Cat# 18257). A 1-mlResource Q (Pharmacia) anion-exchange column was used to evaluate themethod developed by Huyghe et al. using their HEPES buffer system. Thismethod was only tried for the Bioreactor system.

The buffers used in the present HPLC system were Buffer A: 10 mM trisbuffer, pH 9.0. Buffer B: 1.5 M NaCl in buffer A, pH 9.0. The bufferswere filtered through a 0.22 μm bottle top filter by Corning (Cat#25970-33). All of the samples were filtered through a 0.8/0.22 μmAcrodisc PF from Gelman Sciences (Cat# 4187) before injection.

The sample is injected onto the HPLC column in a 60-100 μl volume. Afterinjection, the column (TosoHaas) is washed with 20% B for 3 min at aflow rate of 0.75 ml/min. A gradient is then started, in which B isincreased from 20% to 50% over 6 min. Then the gradient is changed from50% to 100% B over 3 min, followed by 100% B for 6 min. The saltconcentration is then changed back stepwise to 20% again over 4 min, andmaintained at 20% B for another 6 min. The retention time of the Adp53is 9.5±0.3 min with A₂₆₀/A₂₈₀≅1.26±0.03. Cleaning of the column aftereach chromatographic run is accomplished by injecting 100 μl of 0.15 MNaOH and then running the gradient.

Example 2 Effect of Medium Perfusion Rate in Cellcube™ on VirusProduction and Purification

For a perfusion cell culture system, such as the Cellcube™, mediumperfusion rate plays an important role on the yield and quality ofproduct. Two different medium perfusion strategies were examined. Onestrategy was to keep the glucose concentration in the Cellcube™ ≧2 g/L(high perfusion rate). The other one was to keep the glucoseconcentration ≧1 g/L (low medium perfusion rate).

No significant changes in the culture parameters, such as pH, DO, wasobserved between the two different perfusion rates. Approximatelyequivalent amount of crude viruses (before purification) were producedafter harvesting using 1% Tween-20 lysis solution as shown in Table 5.However, dramatic difference was seen on the HPLC profiles of the viralsolutions from the high and low medium perfusion rate production runs.TABLE 5 Effect of medium glucose concentration on virus yield Glucoseconcentration (g/L) ≧2.0 ≧1.0 Crude virus yield (PFU) 4 × 10¹² 4.9 ×10¹²

As shown in FIG. 1, a very well separated virus peak (retention time9.39 min) was produced from viral solution using low medium perfusionrate. It was found that virus with adequate purity and biologicalactivity was attained after a single step ion exchange chromatographicpurification of the virus solution produced under low medium perfusionrate. On the other hand, no separated virus peak in the retention timeof 9.39 min was observed from viral solution produced using high mediumperfusion rate. This suggests that contaminants which have the sameelution profile as the virus were produced under high medium perfusionrate. Although the nature of the contaminants is not yet clear, it isexpected that the contaminants are related to the increasedextracellular matrix protein production under high medium perfusion rate(high serum feeding) from the producer cells. This poor separationcharacteristic seen on the HPLC created difficulties for process IECpurification as shown in the following Examples. As a result, mediumperfusion rate used during the cell growth and the virus productionphases in the Cellcube™ has a significant effect on the downstream IECpurification of the virus. Low medium perfusion rate is recommended.This not only produces easy to purify crude product but also offers morecost-effective production due to the reduced medium consumption.

Example 3 Methods of Cell Harvest and Lysis

Based on previous experience, the inventors first evaluated thefreeze-thaw method. Cells were harvested from the Cellcube™ 45-48 hrpost-infection. First, the Cellcube™ was isolated from the culturesystem and the spent medium was drained. Then, 50 mM EDTA solution waspumped into the Cube to detach the cells from the culture surface. Thecell suspension thus obtained was centrifuged at 1,500 rpm (BeckmanGS-6KR) for 10 min. The resultant cell pellet was resuspended inDulbecco's phosphate buffered saline (DPBS). The cell suspension wassubjected to 5 cycles of freeze/thaw between 37° C. water bath anddry-ice ethanol bath to release virus from the cells. The crude celllysate (CCL) thus generated was analyzed on HPLC.

FIG. 2 shows the HPLC profile. No virus peak is observed at retentiontime of 9.32 min. Instead, two peaks at retention times of 9.11 and 9.78min are produced. This profile suggests that the other contaminantshaving similar elution time as the virus exist in the CCL and interferewith the purification of the virus. As a result, very low purificationefficiency was observed when the CCL was purified by IEC using FPLC.

In addition to the low purification efficiency, there was a significantproduct loss during the cell harvest step into the EDTA solution asindicated in Table 6. Approximately 20% of the product was lost into theEDTA solution which was discarded. In addition, about 24% of the crudevirus product is present in the spent medium which was also discarded.Thus, only 56% of the crude virus product is in the CCL. Furthermore,freeze-thaw is a process of great variation and very limitedscaleability. A more efficient cell lysis process with less product lossneeded to be developed. TABLE 6 Loss of virus during EDTA harvest ofcells from Cellcube ™ Waste EDTA Crude product Spent harvest Crude cellTotal crude Medium Solution lysate product (PFU) Volume (ml) 2800 200082 — Titer (PFU/ml) 2.6 × 10⁸  3 × 10⁸    2 × 10¹⁰ — Total virus 7.2 ×10¹¹ 6 × 10¹¹ 1.64 × 10¹² 3 × 10¹² (PFU) Percentage 24% 20% 56%

Data was generated from 1 mer Cellcube™. TABLE 7 Evaluation of non-ionicdetergents for cell lysis Detergents Concentrations (w/v) ChemistryComments Thesit   1% Dodecylpoly(ethylene glycol ether)_(n,) Large 0.5%n = 9-10 Precipitate 0.1% NP-40   1%Ethylphenolpoly(ethylene-glycolether)_(n) Large 0.5% n = 9-11precipitate 0.1% Tween-20   1%Poly(oxyethylene)_(n)-sorbitan-monolaurate Small 0.5% n = 20 precipitate0.1% Brij-58   1% Cetylpoly(ethyleneglycolether)_(n) n = 20 Cloudy 0.5%Solution 0.1% Triton X-100   1% Octylphenolpoly(ethyleneglycolether)_(n)Large 0.5% n = 10 precipitate 0.1%

Detergents have been used to lyse cells to release intracellularorganelles. Consequently, the inventors evaluated the detergent lysismethod for the release of adenovirus. Table 7 lists the 5 differentnon-ionic detergents that were evaluated for cell lysis. Cells wereharvested from the Cellcube™ 48 hr post-infection using 50 mM EDTA. Thecell pellet was resuspended in the different detergents at variousconcentrations listed in Table 7.

Cell lysis was carried out at either room temperature or on ice for 30min. Clear lysis solution was obtained after centrifugation to removethe precipitate and cellular debris. The lysis solutions were treatedwith Benzonase and then analyzed by HPLC. FIG. 3 shows the HPLC profilesof lysis solutions from the different detergents. Thesit and NP-40performed similarly as Triton X-100. Lysis solution generated from 1%Tween-20 gave the best virus resolution with the least virus resolutionbeing observed with Brij-58. More efficient cell lysis was found atdetergent concentration of 1% (w/v). Lysis temperature did notcontribute significantly to the virus resolution under the detergentconcentrations examined. For the purpose of process simplicity, lysis atroom temperature is recommended. Lysis solution composed of 1% Tween-20in 20 mM Tris+0.25M NaCl+1 mM MgCl₂, pH=7.50 was employed for cell lysisand virus harvest in the Cellcube™.

Example 4 Effects of Concentration/Diafiltration on Virus Recovery

Virus solution from the lysis step was clarified and filtered beforeconcentration/diafiltration. TFF membranes of different NMWCs, including100K, 300K, 500K, and 1000K, were evaluated for efficientconcentration/diafiltration. The highest medium flux with minimal virusloss to the filtrate was obtained with a membrane of 300K NMWC. BiggerNMWC membranes offered higher medium flux, but resulted in greater virusloss to the filtrate, while smaller NMWC membranes achieved aninsufficient medium flux. Virus solution was first concentrated 10-fold,which was followed by 4 sample volumes of diafiltration against 20 mMTris+0.25 M NaCl+1 mM MgCl₂, pH=9.00 buffer using the constant volumemethod. During the concentration/diafiltration process, pressure dropacross the membrane was kept ≦5 psi. Consistent, high level virusrecovery was demonstrated during the concentration/diafiltration step asindicated in Table 8. TABLE 8 Concentration/diafiltration of crude virussolution Titer (PFU/ml) Volume (ml) Total virus (PFU) Recovery Run #1Run #2 Run #1 Run #2 Run #1 Run #2 Run #1 Run #2 Before 2.6 × 10⁹   2 ×10⁹ 1900 2000 4.9 × 10¹²   4 × 10¹² conc./diafl. Post 2.5 × 10¹⁰ 1.7 ×10¹⁰ 200 200   5 × 10¹² 3.4 × 10¹² 102% 85% conc./diafl. Conc. 9.5 10Factor Filtrate   5 × 10⁵   1 × 10⁶ 3000 3000 1.5 × 10⁹   3 × 10⁹

Example 5 Effect of Salt Addition on Benzonase Treatment

Virus solution after concentration/diafiltration was treated withBenzonase (nuclease) to reduce the concentration of contaminatingnucleic acid in virus solution. Different working concentrations ofBenzonase, which included 50, 100, 200, 300 units/ml, were evaluated forthe reduction of nucleic acid concentrations. For the purpose of processsimplicity, treatment was carried out at room temperature overnight.Significant reduction in contaminating nucleic acid that is hybridizableto human genomic DNA probe was seen after Benzonase treatment.

Table 9 shows the reduction of nucleic acid concentration before andafter Benzonase treatment. Virus solution was analyzed on HPLC beforeand after Benzonase treatment. As shown in FIG. 4A and FIG. 4B, dramaticreduction in the contaminating nucleic acid peak was observed afterBenzonase treatment. This is in agreement with the result of the nucleicacid hybridization assay. Because of the effectiveness, a Benzonaseconcentration of 100 u/ml was employed for the treatment of the crudevirus solution. TABLE 9 Reduction of contaminating nucleic acidconcentration in virus solution Before After Treatment TreatmentReduction Contaminating 200 μg/ml 10 ng/ml 2 × 10⁴-fold nucleic acidconcentration

-   -   Treatment condition: Benzonase concentration: 100 u/ml,        temperature: room temperature, time: overnight.

Considerable change in the HPLC profile was observed pre- andpost-Benzonase treatment. No separated virus peak was detected atretention time of 9.33 min after Benzonase treatment. At the same time,a major peak with high 260 nm adsorption at retention time of 9.54 minwas developed. Titer assay results indicated that Benzonase treatmentdid not negatively affect the virus titer and virus remained intact andinfectious after Benzonase treatment. It was reasoned that cellularnucleic acid released during the cell lysis step interacted with virusand either formed aggregates with the virus or adsorbed onto the virussurface during Benzonase treatment.

To minimize the possible nucleic acid virus interaction during Benzonasetreatment, different concentrations of NaCl was added into the virussolution before Benzonase treatment. No dramatic change in the HPLCprofile occurred after Benzonase treatment in the presence of 1 M NaClin the virus solution. FIG. 5 shows the HPLC profile of virus solutionafter Benzonase treatment in the presence of 1M NaCl. Unlike that shownin FIG. 4B, virus peak at retention time of 9.35 min still exists postBenzonase treatment. This result indicates that the presence of 1M NaClprevents the interaction of nucleic acid with virus during Benzonasetreatment and facilitates the further purification of virus fromcontaminating nucleic acid.

Example 6 Ion Exchange Chromatographic Purification

The presence of negative charge on the surface of adenovirus atphysiological pH conditions prompted evaluation of anionic ionexchangers for adenovirus purification. The strong anionic ion exchangerToyopearl Super Q 650M was used for the development of a purificationmethod. The effects of NaCl concentration and pH of the loading buffer(buffer A) on virus purification was evaluated using the FPLC system.

A) Method Development

For ion exchange chromatography, buffer pH is one of the most importantparameters and can have dramatic influence on the purificationefficiency. In reference to the medium pH and conductivity used duringvirus production, the inventors formulated 20 mM Tris+1 mM MgCl₂+0.2MNaCl, pH=7.50 as buffer A. A XK16 column packed with Toyopearl SuperQ650M with a height of 5 cm was conditioned with buffer A.

A sample of 5 ml of Benzonase treated concentrated/diafiltrated virussupernatant from the Celligen bioreactor was loaded onto the column.After washing the column, elution was carried out with a linear gradientof over 10 column volumes of buffer B formulation to reach mM Tris+1 mMMgCl₂+2M NaCl, pH=7.50.

FIG. 6 shows the elution profile. Three peaks were observed duringelution without satisfactory separation among them. Control studyperformed with 293 cell conditioned medium (with no virus) showed thatthe first two peaks are virus related. To further improve the separationefficiency, the effect of buffer pH was evaluated. Buffer pH wasincreased to 9.00 while keeping other conditions constant. Much improvedseparation, as shown in FIG. 7, was observed as compared to that ofbuffer pH of 7.50. Fractions #3, #4, and #8 were analyzed on HPLC.

As shown in FIG. 8, the majority of virus was found in fraction #4, withno virus being detected in fractions #3 and #8. Fraction #8 was found tobe mainly composed of contaminating nucleic acid. However, thepurification was still not optimal. There is overlap between fractions#3 and #4 with contaminants still detected in fraction #4.

Based on the chromatogram in FIG. 7, it was inferred that furtherimprovement of virus purification could be achieved by increasing thesalt concentration in buffer A. As a result, the contaminants present inthe fraction #3, which is prior to the virus peak, can be shifted to theflow through faction. The NaCl concentration in buffer A was increasedto 0.3 M while keeping other conditions constant. FIG. 9 shows theelution profile under the condition of 0.3 M NaCl in buffer A.

Dramatic improvement in purification efficiency was achieved. Asexpected the contaminant peak observed in FIG. 7 was eliminated underthe increased salt condition. Samples from crude virus sup, flowthrough, peak #1, and peak #2 were analyzed on HPLC and the results areshown in FIG. 10. No (Virus was detected in the flow through fraction.The majority of the contaminants present in the crude material werefound in the flow through. HPLC analysis of peak #1 showed a single welldefined virus peak. This HPLC profile is equivalent to that obtainedfrom double CsCl gradient purified virus. Peaks observed at retentiontimes of 3.14 and 3.61 min in CsCl gradient purified virus are glycerolrelated peaks. The purified virus has a A260/A280 ratio of 1.27±0.03.This similar to the value of double CsCl gradient purified virus as wellas the results reported by Huyghe et al. (1996). Peak #2 is composedmainly of contaminating nucleic acid. Based on the purification result,the inventors proposed the following method for IEC purification ofadenovirus sup from the bioreactor.

-   -   Buffer A: 20 mM Tris+1 mM MgCl₂+0.3M NaCl, pH=9.00    -   Buffer B: 20 mM Tris+1 mM MgCl₂+2M NaCl, pH=9.00    -   Elution: 10 column volume linear gradient

B) Method Scale-Up

Following the development of the method, purification was scaled-up fromthe XK16 column (1.6 cm I.D.) to a XK50 column (5 cm I.D., 10-foldscale-up) using the same purification method. A similar elution profilewas achieved on the XK50 column as shown in FIG. 11. The virus fractionwas analyzed on HPLC, which indicated equivalent virus purity to thatobtained from the XK16 column.

During the scale-up studies, it was found that it was more convenientand consistent to use conductivity to quantify the salt concentration inbuffer A. The optimal conductivity of buffer A is in the range of 25±2mS/cm at approximately room temperature (21° C.). Samples producedduring the purification process together with double CsCl purified viruswere analyzed on SDS-PAGE.

As shown in FIG. 12, all the major adenovirus structure proteins aredetected on the SDS-PAGE. The IEC purified virus shows equivalentstaining as that of the double CsCl purified virus. Significantreduction in bovine serum albumin (BSA) concentration was achievedduring purification. The BSA concentration in the purified virus wasbelow the detection level of the western blot assay as shown in FIG. 13.

The reduction of contaminating nucleic acid concentration in virussolution during the purification process was determined using nucleicacid slot blot. ³²P labeled human genomic DNA was used as thehybridization probe (because 293 cells are human embryonic kidneycells). Table 10 shows the nucleic acid concentration at differentstages of the purification process. Nucleic acid concentration in thefinal purified virus solution was reduced to 60 pg/ml, an approximate3.6×10⁶-fold reduction compared to the initial virus supernatant. Virustiter and infectious to total particle ratio were determined for thepurified virus and the results were compared to that from double CsClpurification in Table 9. Both virus recovery and particle/PFU ratio arevery similar between the two purification methods. The titer of thecolumn purified virus solution can be further increased by performing aconcentration step. TABLE 10 Removal of contaminating nucleic acidsduring purification Contaminating nucleic acid Steps during purificationconcentration Virus supernatant from bioreactor 220 μg/mlConcentrated/diafiltrated sup 190 μg/ml Sup post Benzonase treatment(O/N, RT,  10 ng/ml 100 u/ml) Purified virus from column 210 pg/mlPurified virus post  60 pg/ml concentration/diafiltration CsCl purifiedvirus 800 pg/ml

Example 7 Other Purification Methods

In addition to the strong anionic ion exchange chromatography, othermodes of chromatographic methods, were also evaluated for thepurification of AdCMVp53 virus (e.g. size exclusion chromatography,hydrophobic interaction chromatography, cation exchange chromatography,or metal ion affinity chromatography). Compared to the Toyopearl SuperQ, all those modes of purification offered much less efficientpurification with low product recovery. Therefore, Toyopearl Super Qresin is recommended for the purification of AdCMVp53. However, otherquaternary ammonium chemistry based strong anionic exchangers are likelyto be suitable for the purification of AdCMVp53 with some processmodifications.

Example 8 Purification of Crude AdCMVp53 virus Generated from Cellcube™

Two different production methods were developed to produce AdCMVp53virus. One was based on microcarrier culture in a stirred tankbioreactor. The other was based on a Cellcube™ bioreactor. As describedabove, the purification method was developed using crude virussupernatant generated from the stirred tank bioreactor. It was realizedthat although the same medium, cells and viruses were used for virusproduction in both the bioreactor and the Cellcube™, the culture surfaceonto which cells attached was different.

In the bioreactor, cells were grown on a glass coated microcarrier,while in the Cellcube™ cells were grown on proprietary treatedpolystyrene culture surface. Constant medium perfusion was used in theCellcube™, on the other hand, no medium perfusion was used in thebioreactor. In the Cellcube™, the crude virus product was harvested inthe form of virally infected cells, which is different from the virussupernatant harvested from the bioreactor.

Crude cell lysate (CCL), produced after 5 cycles freeze-thaw of theharvested virally infected cells, was purified by IEC using the abovedescribed method. Unlike the virus supernatant from the bioreactor, nosatisfactory purification was achieved for the CCL material generatedfrom the Cellcube™. FIG. 14 shows the chromatogram. The result suggeststhat crude virus solution generated from the Cellcube™ by freeze-thawingharvested cells is not readily purified by the IEC method.

Other purification methods, including hydrophobic interaction and metalchelate chromatography, were examined for the purification of virus inCCL. Unfortunately, no improvement in purification was observed byeither method. Considering the difficulties of purification of virus inCCL and the disadvantages associated with a freeze-thaw step in theproduction process, the inventors decided to explore other cell lysismethods.

A) Purification of Crude Virus Solution in Lysis Buffer

As described in Examples 1 and 3, HPLC analysis was used to screendifferent detergent lysis methods. Based on the HPLC results, 1%Tween-20 in 20 mM Tris+0.25 M NaCl+1 mM MgCl₂, pH=7.50 buffer wasemployed as the lysis buffer. At the end of the virus production phase,instead of harvesting the infected cells, the lysis buffer was pumpedinto the Cellcube™ after draining the spent medium. Cells were lysed andvirus released into the lysis buffer by incubating for 30 min.

After clarification and filtration, the virus solution wasconcentrated/diafiltrated and treated with Benzonase to reduce thecontaminating nucleic acid concentration. The treated virus solution waspurified by the method developed above using Toyopearl SuperQ resin.Satisfactory separation, similar to that obtained using virussupernatant from the bioreactor, was achieved during elution. FIG. 15shows the elution profile. However, when the virus fraction was analyzedon HPLC, another peak in addition to the virus peak was detected. Theresult is shown in FIG. 16A.

To further purify the virus, the collected virus fraction wasre-purified using the same method. As shown in FIG. 16B, purity of thevirus fraction improved considerably after the second purification.Metal chelate chromatography was also evaluated as a candidate for thesecond purification. Similar improvement in virus purity as seen withthe second IEC was achieved. However, because of its simplicity, IEC ispreferred as the method of choice for the second purification.

As described above in Example 2, medium perfusion rate employed duringthe cell growth and virus production phases has a considerable impact onthe HPLC separation profile of the Tween-20 crude virus harvest. Forcrude virus solution produced under high medium perfusion rate, two ionexchange columns are required to achieve the required virus purity.

Based on the much improved separation observed on HPLC for virussolution produced under low medium perfusion rate, it is likely thatpurification through one ion exchange column may achieve the requiredvirus purity. FIG. 17 shows the elution profile using crude virussolution produced under low medium perfusion rate. A sharp virus peakwas attained during elution. HPLC analysis of the virus fractionindicates virus purity equivalent to that of CsCl gradient purifiedvirus after one ion exchange chromatography step. FIG. 18 shows the HPLCanalysis result.

The purified virus was further analyzed by SDS-PAGE, western blot forBSA, and nucleic acid slot blot to determine the contaminating nucleicacid concentration. The analysis results are given in FIG. 19A, FIG. 19Band FIG. 19C, respectively. All those analyses indicate that the columnpurified virus has equivalent purity compared to the double CsClgradient purified virus. Table 11 shows the virus titer and recoverybefore and after the column purification. For comparison purposes, thetypical virus recovery achieved by double CsCl gradient purification wasalso included. Similar virus recoveries were achieved by both methods.TABLE 11 Comparison of IEC and double CsCl gradient ultracentrifugationpurification of AdCMVp53 from Cellcube ™ Titer (PFU/ml) A260/A280Particle/PFU Recovery IEC 1 × 10¹⁰ 1.27 36 63% Ultracentrifugation 2 ×10¹⁰ 1.26 38 60%

A) Resin Capacity Study

The dynamic capacity of the Toyopearl Super Q resin was evaluated forthe purification of the Tween-20 harvested virus solution produced underlow medium perfusion rate. One hundred ml of resin was packed in a XK50column. Different amount of crude virus solution was purified throughthe column using the methods described herein.

Virus breakthrough and purification efficiency were analyzed on HPLC.FIG. 20 shows the HPLC analysis results. At a column loading factorgreater than sample/column volume ratio of 2:1, purity of the virusfraction was reduced. Contaminants co-eluted with the virus. At aloading factor of greater than 3:1, breakthrough of the virus into theflow through was observed. Therefore, it was proposed that the workingloading capacity of the resin be in the range of sample/column volumeratio of 1:1.

B) Concentration/Diafiltration Post Purification

A concentration/diafiltration step after column purification serves notonly to increase the virus titer, if necessary, but also to exchange tothe buffer system specified for the virus product. A 300K NMWC TFFmembrane was employed for the concentration step. Because of the absenceof proteinacious and nucleic acid contaminants in the purified virus,very high buffer flux was achieved without noticeable pressure dropacross the membrane.

Approximately 100% virus recovery was achieved during this step bychanging the buffer into 20 mM Tris+1 mM MgCl₂+0.15 M NaCl, pH=7.50. Thepurified virus was also successfully buffer exchanged into DPBS duringthe concentration/diafiltration step. The concentration factor can bedetermined by the virus titer that is desired in the final product andthe titer of virus solution eluted from the column. This flexibilitywill help to maintain the consistency of the final purified virusproduct.

C) Evaluation of Defective Adenovirus in the IEC Purified AdCMVp53

Due to the less than 100% packaging efficiency of adenovirus in producercells, some defective adenoviruses generally exist in crude virussolution. Defective viruses do not have DNA packaged inside the viralcapsid and therefore can be separated from intact virus on CsCl gradientultracentrifugation based the density difference. It is likely that itwould be difficult to separate the defective from the intact virusesbased on ion exchange chromatography assuming both viruses have similarsurface chemistry. The presence of excessive amount of defective viruseswill impact the quality of the purified product.

To evaluate the percentage of defective virus particles present, thepurified and concentrated viruses were subjected to isopicnic CsClultracentrifugation. As shown in FIG. 21, a faint band on top of theintact virus band was observed after centrifugation. Both bands wererecovered and dialyzed against 20 mM Tris+1 mM MgCl₂, pH=7.50 buffer toremove CsCl. The dialyzed viruses were analyzed on HPLC and the resultsare shown in FIG. 22. Both viruses show similar retention time. However,the defective virus has a smaller A260/A280 ratio than that of theintact virus. This is indicative of less viral DNA in the defectivevirus.

The peaks seen at retention times between 3.02 to 3.48 min are producedby glycerol which is added to the viruses (10% v/v) before freezing at−70° C. The percentage of the defective virus was less than 1% of thetotal virus. This low percentage of defective virus is unlikely toimpact the total particle to infectious virus (PFU) ratio in thepurified virus product. Both viruses were analyzed by SDS-PAGE (shown inFIG. 19A). Compared to the intact viruses, defective viruses lack theDNA associated core proteins banded at 24 and 48.5 KD. This result is inagreement with the absence of DNA in defective virus.

D) Process Overview of the Production and Purification of AdCMVp53 Virus

Based on the above process development results, the inventors propose aproduction and purification flow chart for AdCMVp53 as shown in FIG. 23.The step and accumulative virus recovery is included with thecorresponding virus yield based on a 1 mer Cellcube™. The final virusrecovery is about 70±10%. This is about 3-fold higher than the virusrecovery reported by Huyghe et al. (1996) using a DEAE ion exchanger anda metal chelate chromatographic purification procedure for thepurification of p53 protein encoding adenovirus. Approximately 3×10¹²PFU of final purified virus product was produced from a 1 mer Cellcube™.This represents a similar final product yield compared to the currentproduction method using double CsCl gradient ultracentrifugation forpurification.

E) Scale-Up

Successful scale-up studies are have been performed with the 4 merCellcube™ system, and are currently underway to evaluate virusproduction in the 16 mer Cellcube™ system. The crude virus solutionproduced will be filtered, concentrated and diafiltrated using a biggerPellicon cassette. The quality and recovery of the virus will bedetermined. After Benzonase treatment, the crude virus solution will bepurified using a 20 cm and a 30 cm BioProcess column for the 4 mer and16 mer, respectively.

Example 9 Improved Ad-p53 Production in Serum-Free Suspension Culture

Adaptation of 293 cells

293 cells were adapted to a commercially available IS293 serum-freemedia (Irvine Scientific; Santa Ana, Calif.) by sequentially loweringdown the FBS concentration in T-flasks. The frozen cells in one vial ofPDWB were thawed and placed in 10% FBS DMEM media in T-75 flask and thecells were adapted to serum-free IS 293 media in T-flasks by loweringdown the FBS concentration in the media sequentially. After 6 passagesin T-75 flasks the FBS % was estimated to be about 0.019%. The cellswere subcultured two more times in the T flasks before they weretransferred to spinner flasks.

Serum-Free Adapted 293 Cells in T Flasks were Adapted to SuspensionCulture

The above serum-free adapted cells in T-flasks were transferred to aserum-free 250 mL spinner suspension culture (100 mL working volume) forthe suspension culture. The initial cell density was 1.18E+5 vc/mL.During the cell culture the viability decreased and the big clumps ofcells were observed. After 2 more passages in T-flasks the adaptation tosuspension culture was tried again. In a second attempt the media wassupplemented with heparin, at a concentration of 100 mg/L, to preventaggregation of cells and the initial cell density was increased to5.22E+5 vc/mL. During the cell culture there was some increase of celldensity and cell viability was maintained. Afterwards the cells weresubcultured in the spinner flasks for 7 more passages and during thepassages the doubling time of the cells was progressively reduced and atthe end of seven passages it was about 1.3 day which is comparable to1.2 day of the cells in 10% FBS media in the attached cell culture. Inthe serum-free IS 293 media supplemented with heparin almost all thecells existed as individual cells not forming aggregates of cells in thesuspension culture (Table 12). TABLE 12 Serum-Free Suspension Culture:Adaptation to Suspension Passage No. Flask No. Average Doubling Time(days) 11 Viability decreased 13 3.4 14 3.2 15 1 Viability decreasedheparin added 2 4.7 3 5.0 4 3.1 16 1 5.5 2 4.8 3 4.3 4 4.3 17 1 2.9 23.5 3 2.4 4 1.7 18 1 3.5 2 13.1  3 6.1 4 3.8 19 1 2.5 2 2.6 3 2.3 4 2.520 1 1.3 (97% viability) 2 1.5 (99% viability) 3 1.8 (92% viability) 41.3 (96% viability)Viral Production and Growth of Cells in Serum-Free Suspension Culture inSpinner Flask

To test the production of Ad5-CMVp53 vectors in the serum-freesuspension culture the above cells adapted to the serum-free suspensionculture were grown in 100 mL serum-free IS293 media supplemented with0.1% Pluronic F-68 and Heparin (100 mg/L) in 250 mL spinner flasks. thecells were infected at 5 MOI when the cells reached 1.36E+06 viablecells/mL on day 3. The supernatant was analyzed everyday for HPLC viralparticles/mL after the infection. No viruses were detected other thanday 3 sample. On day 3 it was 2.2E+09 vps/mL. The pfu/mL on day 6 was2.6+/−0.6E+07 pfu/mL. The per cell pfu production was estimated to be 19which is approximately 46 times below the attached culture in theserum-supplemented media. As a control the growth of cells was checkedin the absence of an infection. TABLE 13 Serum-Free Suspension Culture:Viral Production and Cell Growth Viral Viral Control infection infectionw/o viral w/o media w/media infection exchange exchange Initial Density(vc/mL) 2.1 × 10⁵ 2.1 × 10⁵ 2.1 × 10⁵ Cell Density at infection 9.1 ×10⁵ 1.4 × 10⁶ 1.5 × 10⁶ (vc/mL) Volumetric viral production NA 2.6 × 10⁷2.8 × 10⁸ (pfu/mL) 6 days P.I. Volumetric viral production NA NA  1.3 ×10¹⁰ (HPLC vps/mL) 6 days P.I. Per cell viral production NA NA 1.3 × 10⁴(HPLC vps/cell)Preparation of Serum-Free Suspension Adapted 293 Cell Banks

As described above, after it was demonstrated the cells produce theAd-p53 vectors, the cells were propagated in the serum-free IS293 mediawith 0.1% F-68 and 100 mg/L heparin in the spinner flasks to makeserum-free suspension adapted cell banks which contain 1.0E+07 viablecells/mL/vial. To collect the cells they were centrifuged down when theywere at mid-log phase growth and the viability was over 90% andresuspended in the serum-free, supplemented IS293 media and centrifugeddown again to wash out the cells. Then the cells were resuspended againin the cryopreservation media which is cold IS293 with 0.1% F-68, 100mg/L heparin, 10% DMSO and 0.1% methylcellulose resulting in 1E+07viable cells/mL. The cell suspension was transferred to sterilecryopreservation vials and they were sealed and frozen in cryocontainerat −70 C overnight. The vials were transferred to liquid nitrogenstorage. The mycoplasma test was negative.

To revive the frozen cells one vial was thawed into the 50 mL serum-freeIS293 media with 0.1% F-68 and 100 mg/L heparin in a T-150. Since thenthe cultures were subcultured three times in 250 mL spinner flasks. Inthe other study one vial was thawed into 100 mL serum-free, supplementedIS293 media in a 250 mL spinner flask. Since then these were subculturedin serum-free spinner flasks 2 times. In both of the studies the cellsgrew very well.

Media Replacement and Viral Production in Serum-Free Suspension Culturein Spinner Flask

In the previous serum-free viral production in the suspension culture inthe spinner flask the per cell viral production was too low for theserum-free suspension production to be practical. It was supposed thatthis might be due to the depletion of nutrients and/or the production ofinhibitory byproducts. To replace the spent media with fresh serum-free,supplemented IS293 media the cells were centrifuged down on day 3 andresuspended in a fresh serum-free IS-293 medium supplemented with F-68and heparin (100 mg/L) and the resulting cell density was 1.20E+06 vc/mLand the cells were infected with Ad5-CMVp53 vectors at 5 MOI. Theextracellular HPLC vps/mL was 7.7E+09 vps/mL on day 3, 1.18E+10 vps/mLon day 4, 1.2E+10 vps/mL on day 5 and 1.3E+10 vps/mL on day 6 and thepfu/mL on day 6 was 2.75+/−0.86E+08 tvps/mL. The ratio of HPLC viralparticles to pfus was about 47. Also the cells have been centrifugeddown and lysed with the same type of the detergent lysis buffer as usedin the harvest of CellCube. The cellular HPLC vps/mL was 1.6E+10 vps/mLon day 2, 6.8E+09 vps/mL on day 3, 2.2E+09 vps/mL on day 4, 2.24E+09vps/mL on day 5 and 2.24E+09 vps/mL on day 6.

The replacement of the spent media with a fresh serum-free, supplementedIS 293 media resulted in the significant increase in the production ofAd-p53 vectors. The media replacement increased the production ofextracellular HPLC viral particles 3.6 times higher above the previouslevel on day 3 and the production of extracellular pfu titer ten timeshigher above the previous level on day 6. Per cell production of Ad-p53vectors was estimated to be approximately 1.33E+04 HPLC vps.

The intracellular HPLC viral particles peaked on day 2 following theinfection and then the particle numbers decreased. In return theextracellular viral particles increased progressively to the day 6 ofharvest. Almost all the Ad-p53 vectors were produced for the 2 daysfollowing the infection and intracellularly localized and then theviruses were released outside of the cells. Almost half of the viruseswere released outside of the cells into the supernatant between day 2and day 3 following the infection and the rate of release decreased astime goes on.

All the cells infected with Ad-p53 vectors lost their viability at theend of 6 days after the infection while the cells in the absence ofinfection was 97% viable. In the presence of infection the pH of thespent media without the media exchange and with the media exchange was6.04 and 5.97, respectively, while the one in the absence of theinfection was 7.00 (Table 12).

Viral Production and Cell Culture in Stirred Bioreactor with MediaReplacement and Gas Overlay

To increase the production of Ad-p53 vectors, a 5 L CelliGen bioreactorwas used to provide a more controlled environment. In the 5 L CelliGenbioreactor the pH and the dissolved oxygen as well as the temperaturewas controlled. Oxygen and carbon dioxide gas was connected to thesolenoid valve for oxygen supply and the pH adjustment, respectively.For a better mixing while generating low shear environment amarine-blade impeller was implemented. Air was supplied all the timeduring the operation to keep a positive pressure inside the bioreactor.

To inoculate the bioreactor a vial of cells was thawed into 100 mLserum-free media in a 250 mL spinner flask and the cells were expandedin 250 or 500 mL spinner flasks. 800 mL cell inoculum, grown in 500 mLflasks, was mixed with 2700 mL fresh media in a 10 L carboy andtransferred to the CelliGen bioreactor by gas pressure. The initialworking volume of the CelliGen bioreactor was about 3.5 L culture. Theagitation speed of the marine-blade impeller was set at 80 rpm, thetemperature at 37° C., pH at 7.1 at the beginning and 7.0 after theinfection and the DO at 40% all the time during the run.

The initial cell density was 4.3E+5 vc/mL (97% viability) and 4 dayslater when the cell density reached to 2.7E+6 vc/mL (93% viability) thecells were centrifuged down and the cells were resuspended in a freshmedia and transferred to the CelliGen bioreactor. After the mediaexchange the cell density was 2.1E+6 vc/mL and the cells were infectedat MOI of 10. Since then the DO dropped to below 40%. To keep the DOabove 40%, about 500 mL of culture was withdrawn from the CelliGenbioreactor to lower down the oxygen demand by the cell culture and theupper marine-blade was positioned close to the interface between the gasand the liquid phase to improve the oxygen transfer by increasing thesurface renewal. Since then the DO could be maintained above 40% untilthe end of the run.

For pH control, CO₂ gas was used to acidify the cell culture and 1 NNaHCO₃ solution to make the cell culture alkaline. The pH control wasinitially set at 7.10. The initial pH of the cell culture was about pH7.41. Approximately 280 mL 1N NaHCO₃ solution was consumed until the pHof cell culture stabilized around pH 7.1. After the viral infection ofthe cell culture, the pH control was lowered down to pH 7.0 and the CO₂gas supply line was closed off to reduce the consumption of NaHCO₃solution. The consumption of too much NaHCO₃ solution for pH adjustmentwould increase the cell culture volume undesirably. Since then 70 mL 1NNaHCO₃ solution was consumed and the pH was in the range between 7.0 and7.1 most of the time during the run. The temperature was controlledbetween 35° C. and 37° C.

After the infection the viability of the cells decreased steadily untilday 6 of harvest after the infection. On the harvest day none of thecells was viable. The volumetric viral production of the CelliGenbioreactor was 5.1E+10 HPLC vps/mL compared to the 1.3E+10 vps/mL in thespinner flask. The controlled environment in the CelliGen bioreactorincreased the production of Ad-p53 vectors 4-fold compared to thespinner flasks with media replacement. This is both due to the increaseof the cell density at the time of infection from 1.2E+6 to 2.1E+6 vc/mLand the increase of per cell viral production from 1.3E+4 to 2.5E+4vps/mL. The 2.5E+4 vps/mL is comparable to the 3.5E+4 vps/cell in theserum-supplemented, attached cell culture.

Viral Production and Cell Culture in Stirred and Sparged Bioreactor

In the first study the cells were successfully grown in an stirredbioreactor for viral production, and the oxygen and CO₂ were supplied bygas overlay in the headspace of a bioreactor. However, this method willlimit the scale-up of the cell culture system because of its inefficientgas transfer. Therefore in the second study, to test the feasibility ofthe scale up of the serum-free suspension culture and investigate thegrowth of cells and Ad-p53 production in a sparged bioreactor, pureoxygen and CO₂ gases were supplied by bubbling through the serum-freeIS293 media supplemented with F-68 (0.1%) and heparin (100 mg/L).

Pure oxygen was bubbled through the liquid media to supply the dissolvedoxygen to the cells and the supply of pure oxygen was controlled by asolenoid valve to keep the dissolved oxygen above 40%. For efficientoxygen supply while minimizing the damage to the cells a stainless steelsintered air diffuser, with a nominal pore size of which isapproximately 0.22 micrometer, was used for the pure oxygen delivery.The CO₂ gas was also supplied to the liquid media by bubbling from thesame diffuser and tube as the pure oxygen to maintain the pH around 7.0.For pH control Na₂CO₃ solution (106 g/L) was also hooked up to thebioreactor. Air was supplied to the head space of the bioreactor to keepa positive pressure inside the bioreactor. Other bioreactorconfiguration was the same as the first study.

Inoculum cells were developed from a frozen vial. One vial of frozencells (1.0E+7 vc) was thawed into 50 mL media in a T-150 flask andsubcultured 3 times in 200 mL media in 500 mL spinner flasks. 400 mL ofinoculum cells grown in 2 of 500 mL spinner flasks were mixed with IS293media with F-68 and heparin in a 10 L carboy to make 3.5 L cellsuspension and it was transferred to the 5 L CelliGen bioreactor.

The initial cell density in the bioreactor was 3.0E+4 vc/mL. The initialcell density is lower than the first study. In the first study four of500 mL spinner flasks were used as the inoculum. Even with the lowerinitial cell density the cells were grown up to 1.8E+6 vc/mL on day 7 inthe sparged environment and the viability was 98%. During the 7 days'growth, glucose concentration decreased from 5.4 g/L to 3.0 g/L andlactate increased from 0.3 g/L to 1.8 g/L.

On day 7, when the cell density reached 1.8E+6 vc/mL, the cells in thebioreactor were centrifuged down and resuspended in 3.5 L freshserum-free IS293 media with F-68 and heparin in a 10 L carboy. The 293cells were infected with 1.25E+11 pfu Ad-p53 and transferred to theCelliGen bioreactor. In the bioreactor, cell viability was 100% but thecell density was only 7.2E+5 vc/mL. There was a loss of cells during themedia exchange operation. The viral titer in the media was measured as2.5E+10 HPLC vps/mL on day 2, 2.0E+10 on day 3, 2.8E+10 on day 4,3.5E+10 on day 5 and 3.9E+10 HPLC vps/mL on day 6 of harvest. The firstCelliGen bioreactor study with gas overlay produced 5.1E+10 HPLC vps/mL.The lower virus concentration in the second run was likely due to thelower cell density at the time of infection. Compared to the 7.2E+5vc/mL in the second run, 2.1 E+6 vc/mL was used in the first run.Actually the per cell production of Ad-p53 in the second spargedCelliGen bioreactor is estimated to be 5.4E+4 vps/cell which is thehighest per cell production ever achieved so far. The per cellproduction in the first serum-free CellGen bioreactor without spargingand the serum-supplemented T-flask was 2.5E+4 vps/cell and 3.5E+4vps/cell, respectively.

After the viral infection, the viability of the cells decreased from100% to 13% on day 6 of harvest. During those 6 days after the infectionthe glucose concentration decreased from 5.0 g/L to 2.1 g/L and thelactate increased from 0.3 g/L to 2.9 g/L. During the entire period ofoperation about 20 mL of Na₂CO₃ (106 g/L) solution was consumed.

The experimental result shows that it is technically and economicallyfeasible to produce Ad-p53 in the sparged and stirred bioreactor.Scale-up and large-scale unit operation of sparged and stirredbioreactor are well established.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1.-69. (canceled)
 70. A method for producing a purified adeno-associatedvirus composition comprising: a) growing host cells; b) providingnutrients to the host cells by perfusion or through a fed-batch process;c) infecting the host cells with an adeno-associated virus; d) lysingthe host cells to provide a cell lysate comprising adeno-associatedvirus; and e) purifying adeno-associated virus from the lysate toprovide a purified adeno-associated virus composition.
 71. The method ofclaim 70, further comprising infecting the host cells with a helpervirus.
 72. The method of claim 71, wherein the helper virus isadenovirus.
 73. The method of claim 70, further comprising subjectingthe lysate to a clarification step.
 74. The method of claim 70, furthercomprising subjecting the lysate to a concentration step.
 75. The methodof claim 70, further comprising subjecting the lysate to a diafiltrationstep.
 76. The method of claim 70, further comprising treating the lysatewith a nuclease.
 77. The method of claim 76, wherein the nuclease isBENZONASE™.
 78. The method of claim 76, wherein the nuclease isPULMOZYME™.
 79. The method of claim 70, wherein purifying theadeno-associated virus from the lysate comprises using at least onechromatography step.
 80. The method of claim 79, wherein purifying theadeno-associated virus from the lysate comprises using at least twochromatography steps.
 81. The method of claim 80, wherein purifying theadeno-associated virus from the lysate comprises using at least threechromatography steps.